EP4679085A1 - Biomarker and use thereof - Google Patents

Biomarker and use thereof

Info

Publication number
EP4679085A1
EP4679085A1 EP24770772.2A EP24770772A EP4679085A1 EP 4679085 A1 EP4679085 A1 EP 4679085A1 EP 24770772 A EP24770772 A EP 24770772A EP 4679085 A1 EP4679085 A1 EP 4679085A1
Authority
EP
European Patent Office
Prior art keywords
biomarker
subject
barrier function
specimen
intestinal barrier
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24770772.2A
Other languages
German (de)
French (fr)
Inventor
Miyabi YASUDA
Masashi Morifuji
Saori Takahashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Meiji Co Ltd
Original Assignee
Meiji Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Meiji Co Ltd filed Critical Meiji Co Ltd
Publication of EP4679085A1 publication Critical patent/EP4679085A1/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a biomarker and use thereof.
  • the present invention relates to a method for evaluating intestinal barrier function in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • the present invention also relates to a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the diagnosis, use of a biomarker for the diagnosis, a kit for the diagnosis, and a computer program for allowing a computer to execute the method.
  • the present invention also relates to a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • the present invention also relates to a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • the present invention also relates to a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the acquisition, use of a biomarker for the acquisition, a kit for the acquisition, and a computer program for allowing a computer to execute the method.
  • the present invention also relates to a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the screening, use of a biomarker for the screening, a kit for the screening, and a computer program for allowing a computer to execute the method.
  • the present invention also relates to a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • the present invention also relates to a method for producing a food product or pharmaceutical composition.
  • the intestinal lumen is a space surrounded by a single layer of mucosal epithelial cells, and the intestinal lumen (the outside of the living body) and the inside of the living body are partitioned from each other via mucosal epithelial cells.
  • the intestinal tract Exposed to foreign bodies (e.g., harmful microorganisms, toxins, allergens) incorporated from the ambient environment into the intestinal lumen, the intestinal tract is an important organ in terms not only of absorption of nutrients but also of defense.
  • the intestinal barrier function is a function to prevent foreign bodies (e.g., harmful microorganisms, toxins, allergens) incorporated from the ambient environment into the intestinal lumen from intruding into the living body.
  • the intestinal barrier function involves a physical barrier and a chemical barrier.
  • the physical barrier is a barrier that serves literally as a physical obstacle to prevent the intrusion of foreign bodies.
  • the physical barrier includes a mucus layer covering intestinal epithelial cells, glycocalyx, which is an assembly of sugar chains present on the surfaces of intestinal epithelial cells, and a tight junction structure, which is a cell-cell adhesion apparatus for intestinal epithelial cells.
  • the chemical barrier is a molecular group that inhibits foreign body intrusion by exerting antibacterial activity, neutralization activity, and the like through causing chemical change to foreign bodies.
  • the molecular group includes antibacterial peptides (e.g., defensin family molecules, Reg3 family molecules, lactoferrin, lysozyme) produced from intestinal epithelial cells such as Paneth cells.
  • antibacterial peptides e.g., defensin family molecules, Reg3 family molecules, lactoferrin, lysozyme
  • intestinal epithelial cells such as Paneth cells.
  • M cells which incorporate an antigen and present it to dendritic cells
  • IgA immunoglobulin A
  • Reduction in intestinal barrier function causes various diseases or symptoms (e.g., enteritis, allergic diseases, psychiatric diseases, non-alcoholic fatty liver diseases, type II diabetes mellitus, metabolic syndrome, adiposity).
  • diseases or symptoms e.g., enteritis, allergic diseases, psychiatric diseases, non-alcoholic fatty liver diseases, type II diabetes mellitus, metabolic syndrome, adiposity.
  • Lactulose/mannitol test (sugar tolerance test) is known as a testing method for intestinal barrier function. Intestinal permeability is tested in the lactulose/mannitol test, and increase in intestinal permeability is indicative of deterioration of intestinal barrier function (in particular, physical barrier functions including the tight junction structure).
  • the lactulose/mannitol test has drawbacks including forcing test subjects to have long confinement time and fasting time and laying burdens on the digestive tracts of test subjects by sugar alcohol to be ingested.
  • Non Patent Literature 1 Blood zonulin levels
  • Non Patent Literature 2 Blood zonulin levels
  • An object of the present invention is to provide a method for evaluating intestinal barrier function in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the diagnosis, use of a biomarker for the diagnosis, a kit for the diagnosis, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • Another object of the present invention is to provide a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the acquisition, use of a biomarker for the acquisition, a kit for the acquisition, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the screening, use of a biomarker for the screening, a kit for the screening, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for producing a food product or a pharmaceutical composition.
  • the substance group consisting of the substances listed in Table 1 is referred to as "the substance group of the present invention".
  • Table 1 (Substance group of present invention) No. Substance name 1 Melamine 2 Azelaic acid 3 Urocanic acid 4 Acetanilide 5 Paraxanthine 6 4-Acetamidobutanoic acid 7 Glycerol 8 N-Acetylputrescine 9 N8-Acetylspermidine 10 Threonine 11 Aminophylline 12 2-Oxooctanoic acid 13 Cyclohexanecarboxylic acid 14 Muscimol 15 3-Methoxytyrosine 16 Theobromine 17 Methionine sulfoxide 18 Glyoxylic acid 19 Serotonin 20 Histidylglutamic acid (His-Glu) 21 Guanidinosuccinic acid 22 Uracil 23 Ergothioneine 24 Betonicine 25 Adenine 26 N-Methylnicotinamide 27 4-Hexy
  • the present invention encompasses the following inventions.
  • the present invention provides a method for evaluating intestinal barrier function in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • the present invention also provides a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the diagnosis, use of a biomarker for the diagnosis, a kit for the diagnosis, and a computer program for allowing a computer to execute the method.
  • the present invention also provides a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • the present invention also provides a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • the present invention also provides a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the acquisition, use of a biomarker for the acquisition, a kit for the acquisition, and a computer program for allowing a computer to execute the method.
  • the present invention also provides a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the screening, use of a biomarker for the screening, a kit for the screening, and a computer program for allowing a computer to execute the method.
  • the present invention also provides a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • the present invention also provides a method for producing a food product or pharmaceutical composition.
  • E-X indicates ⁇ 10 -X
  • E+Y indicates ⁇ 10 Y .
  • Aspect 1 of the present invention relates to a method for evaluating intestinal barrier function in a subject.
  • a biomarker in a specimen derived from the subject is used as an indicator for evaluating intestinal barrier function in the subject.
  • the subject may be any animal that has blood without limitation.
  • Such animals include vertebrates including mammals, reptiles, birds, amphibians, and fish, mammals and birds are preferred, and mammals are more preferred.
  • the mammals include primates (e.g., humans, gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), domestic animals (e.g., cattle, pigs, sheep, goats, horses), and pet animals (e.g., dogs, cats), and humans are preferred.
  • the birds include poultry (e.g., chickens, wild ducks, ducks).
  • Blood derived from the subject can be obtained from the subject according to a conventional method, and the obtained blood can be used as a specimen.
  • Serum and plasma derived from the subject can be obtained from blood obtained from the subject according to a conventional method, and the obtained serum and plasma can each be used as a specimen.
  • the blood may be blood immediately after being obtained from the subject, or blood obtained by thawing blood that have been cryopreserved immediately after being obtained from the subject.
  • the serum and plasma may, respectively, be serum and plasma immediately after being obtained from blood, or serum and plasma obtained by thawing serum and plasma that have been cryopreserved immediately after being obtained from blood.
  • the temperature for the cryopreservation is preferably -100°C or more and -10°C or less, more preferably -80°C or more and -15°C or less, and even more preferably -80°C or more and -60°C or less.
  • the cryopreservation can be performed for blood, serum, or plasma with the blood, serum, or plasma contained in a container.
  • the period of the cryopreservation is preferably 180 days or less, more preferably 60 days or less, and even more preferably 30 days or less from blood, serum, or plasma sampling.
  • blood, serum, or plasma within 180 days from blood, serum, or plasma sampling is preferably subjected to measurement of a biomarker
  • blood, serum, or plasma within 60 days from blood, serum, or plasma sampling is more preferably subjected to measurement of a biomarker
  • blood, serum, or plasma within 30 days from blood, serum, or plasma sampling is even more preferably subjected to measurement of a biomarker.
  • the biomarker is a substance or substance group that is present in living bodies and such that the increase or decrease of the in vivo concentration reflects the presence of a specific condition, symptom, disease, or the like, the degree of a symptom, the risk of development, the risk of being affected, a preventing effect, a therapeutic effect, an ameliorating effect, and so on.
  • a substance group is a combination of one or more, preferably two or more substances.
  • the biomarker is a substance or substance group to be measured in the present invention, and used as an indicator for evaluating intestinal barrier function in a subject.
  • the specimen derived from the subject is blood, serum, or plasma.
  • blood refers to whole blood.
  • the specimen derived from the subject is preferably plasma.
  • the biomarker in the specimen derived from the subject includes one or more substances selected from the substance group of the present invention.
  • the biomarker in the specimen derived from the subject may include one substance selected from the substance group of the present invention, or two or more substances selected from the substance group of the present invention.
  • the biomarker in the specimen derived from the subject preferably includes two, three, or four or more substances selected from the substance group of the present invention.
  • intestinal barrier function in the subject can be evaluated with high accuracy by using one or more substances selected from the substance group of the present invention as the biomarker.
  • the substances of No. 1 to No. 15 and No. 27 to No. 40 in the substance group of the present invention give higher accuracy of evaluating intestinal barrier function than other substances in the substance group of the present invention, and the substances of No. 1 to No. 4 and No. 27 to No. 30 in the substance group of the present invention give particularly higher accuracy of evaluating intestinal barrier function than other substances in the substance group of the present invention.
  • the specimen derived from the subject is blood, serum, or plasma
  • the biomarker in the specimen derived from the subject includes one or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide).
  • the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 5 to No. 26 in the substance group of the present invention).
  • the specimen derived from the subject is blood, serum, or plasma
  • the biomarker in the specimen derived from the subject includes one or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL- ⁇ -leucine).
  • the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 31 to No. 42 in the substance group of the present invention).
  • the specimen derived from the subject is blood, serum, or plasma
  • the biomarker in the specimen derived from the subject includes the substance of No. 2 in the substance group of the present invention (azelaic acid).
  • the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1 and No. 3 to No. 26 in the substance group of the present invention).
  • the specimen derived from the subject is blood, serum, or plasma
  • the biomarker in the specimen derived from the subject includes the substance of No. 3 in the substance group of the present invention (urocanic acid).
  • the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1, No. 2, and No. 4 to No. 26 in the substance group of the present invention).
  • the specimen derived from the subject is blood, serum, or plasma
  • the biomarker in the specimen derived from the subject includes the substance of No. 4 in the substance group of the present invention (acetanilide).
  • the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1 to No. 3 and No. 5 to No. 26 in the substance group of the present invention).
  • the specimen derived from the subject is blood, serum, or plasma
  • the biomarker in the specimen derived from the subject includes the substance of No. 5 in the substance group of the present invention (paraxanthine).
  • the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1 to No. 4 and No. 6 to No. 26 in the substance group of the present invention).
  • the method according to Aspect 1 includes the steps of:
  • Step 1a is a step of measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker.
  • the specimen derived from the subject is blood, serum, or plasma.
  • the specimen derived from the subject is blood, serum, or plasma
  • one or more substances selected from the substance group of the present invention is used as a biomarker for evaluating intestinal barrier function in the subject. That is, in the case where the specimen derived from the subject is blood, serum, or plasma, the biomarker to be measured in step 1a includes one or more substances selected from the substance group of the present invention.
  • the biomarker to be measured in step 1a includes one substance selected from the substance group of the present invention
  • the one substance is measured in step 1a.
  • the biomarker to be measured in step 1a includes two or more substances selected from the substance group of the present invention
  • the two or more substances are measured in step 1a.
  • the biomarker to be measured in step 1a includes two, three, or four or more substances selected from the substance group of the present invention (i.e., two, three, or four or more substances selected from the substance group of the present invention are measured in step 1a).
  • measuring one substance means measuring the concentration of the one substance
  • measuring two or more substances means measuring the concentrations of the two or more substances, or determining a combined variable as a statistical analysis value from the concentrations of the two or more substances.
  • biomarker a substance to be subjected to measurement
  • target substance a substance to be subjected to measurement
  • Each concentration may be a relative concentration or an absolute concentration, but is preferably an absolute concentration.
  • the relative concentration of a target substance includes a relative value that correlates with the absolute concentration of a target substance.
  • the relative concentration of a target substance is, for example, a ratio of a measurement value for the target substance to a measurement value for an internal standard (e.g., the relative area value CE or relative area value LC described later).
  • the relative concentration of a target substance may be a ratio given by one measurement sample, or the mean of ratios given by two or more measurement samples. Here, the two or more measurement samples are prepared from the same specimen.
  • DL means a mixture of a D-form and an L-form.
  • Examples of the method for measuring a target substance include liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), gas chromatography-mass spectrometry (GC-MS), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), and CE-MS or LC-MS is preferred among these.
  • CE-MS is suitable for measurement of water-soluble substances
  • LC-MS is suitable for measurement of liposoluble substances.
  • Particularly preferred among different types of CE-MS is capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS).
  • Particularly preferred among different types of LC-MS is liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • Measurement samples to be used in the method for measuring a target substance can be prepared from the specimen according to a conventional method.
  • Each measurement sample to be used in the method for measuring a target substance may contain an internal standard.
  • the relative concentration of a target substance can be determined, for example, as a ratio of a measurement value for the target substance to a measurement value for an internal standard.
  • the absolute concentration of a target substance can be determined, for example, from the relative concentration of the target substance with use of a pre-prepared calibration curve showing the relationship between absolute concentrations of the target substance and relative concentrations of the target substance.
  • the method for measuring a target substance is CE-MS (preferably CE-FTMS). While measurement samples to be used for CE-MS can be prepared from the specimen according to a conventional method, it is preferred to prepare them from the specimen according to a method described in the Examples. While measurement by CE-MS can be performed under common conditions, it is preferred to perform the measurement under conditions described in the Examples. Each measurement sample to be used for CE-MS may contain an internal standard.
  • the internal standard examples include a compound obtained by labeling a target substance with a stable isotope such as DL-phenyl-D5-alanine, a fatty acid methyl ester (e.g., methyl tetradecanoate), ribitol, and an internal standard (H3304-1002; HMT) from Human Metabolome Technologies, Inc. (HMT).
  • a stable isotope such as DL-phenyl-D5-alanine, a fatty acid methyl ester (e.g., methyl tetradecanoate), ribitol
  • H3304-1002 Human Metabolome Technologies, Inc.
  • the relative concentration of a target substance can be determined, for example, as a ratio of the peak area acquired from the target substance in CE-MS to the peak area acquired from an internal standard in CE-MS (referred to as the "relative area value CE").
  • the relative concentration of a target substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples.
  • the peak area for an internal standard can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the internal standard).
  • the peak area for a target substance can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the target substance).
  • the absolute concentration of a target substance can be determined, for example, from the relative concentration of the target substance with use of a pre-prepared calibration curve showing the relationship between absolute concentrations of the target substance and relative concentrations of the target substance.
  • the method for measuring a target substance is LC-MS (preferably LC-MS/MS). While measurement samples to be used for LC-MS can be prepared from the specimen according to a conventional method, it is preferred to prepare them from the specimen according to a method described in the Examples. While measurement by LC-MS can be performed under common conditions, it is preferred to perform the measurement under conditions described in the Examples. Each measurement sample to be used for LC-MS may contain an internal standard.
  • the internal standard examples include a compound obtained by labeling a target substance with a stable isotope such as DL-phenyl-D5-alanine, a fatty acid methyl ester (e.g., methyl tetradecanoate), ribitol, and an internal standard (H3304-1002; HMT) from Human Metabolome Technologies, Inc. (HMT).
  • a stable isotope such as DL-phenyl-D5-alanine, a fatty acid methyl ester (e.g., methyl tetradecanoate), ribitol
  • H3304-1002 Human Metabolome Technologies, Inc.
  • the relative concentration of a target substance can be determined, for example, as a ratio of the peak area acquired from the target substance in LC-MS to the peak area acquired from an internal standard in LC-MS (hereinafter, referred to as the "relative area value LC").
  • the relative concentration of a target substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • the peak area for an internal standard can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the internal standard).
  • the peak area for a target substance can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the target substance).
  • the absolute concentration of a target substance can be determined, for example, from the relative concentration of the target substance with use of a pre-prepared calibration curve showing the relationship between absolute concentrations of the target substance and relative concentrations of the target substance.
  • Measurement of the substances of No. 1 to No. 26 in the substance group of the present invention is preferably performed by CE-MS (in particular, CE-FTMS), and measurement of the substances of No. 27 to No. 42 in the substance group of the present invention is preferably performed by LC-MS (in particular, LC-MS/MS).
  • LC-MS in particular, LC-MS/MS
  • the m/z value of each substance is as shown in Table 9.
  • each "concentration” may be a relative concentration or an absolute concentration, but is preferably an absolute concentration.
  • the relative concentration of each substance is, for example, a relative area value CE.
  • the relative concentration of each substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples.
  • the relative concentration of each substance is, for example, a relative area value LC.
  • the relative concentration of each substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • n may be any integer of 2 or more without limitation, and is, for example, an integer of 3 or more and 42 or less (e.g., 3, 4, 5, 10, 15, 20, 30, 42).
  • the coefficients and constant term depend on the method for measuring substances, the type of concentration of substances (absolute concentration or relative concentration), and the number and types of substances to be combined.
  • the coefficients and constant term can be determined through multinomial logistic regression analysis.
  • the coefficients are partial regression coefficients determined through multinomial logistic regression analysis. Methods for determining the coefficients and constant term through multinomial logistic regression analysis are well known to those skilled in the art. For such methods, reference can be made to documents including Metab Brain Dis, DOI 10.1007/s11011-017-0029-x, Published online: 11 May 2017 , PNAS, October 14, 2003, vol. 100, no. 21, pp.
  • the coefficients to be used in determining the combined variable can be derived from measurement data in such a manner that the combined variable is close to 1 for a group with reduced intestinal barrier function, and close to 0 for a group with non-reduced intestinal barrier function (a group with normal intestinal barrier function).
  • the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by CE-FTMS under conditions described in the Examples, the combined variable can be determined from the concentrations of the two or more substances on the basis of the expression (1), for example, with use of coefficients and a constant term shown in Table 2.
  • the substances of No. 1 to No. 4 in the substance group of the present invention melamine, azelaic acid, urocanic acid, and acetanilide
  • CP_A refers to melamine
  • CP_B refers to azelaic acid
  • CP_C refers to urocanic acid
  • CP_D refers to acetanilide
  • the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL- ⁇ -leucine), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by LC-MS/MS under conditions described in the Examples, the combined variable can be determined from the concentrations of the two or more substances on the basis of the expression (1), for example, with use of coefficients and a constant term shown in Table 3.
  • Step 1b is a step of comparing the measurement value acquired in step 1a with a reference value to acquire a comparison result.
  • a measurement value to be acquired in step 1a is, for example, the relative concentration or absolute concentration of the one substance, and preferably the absolute concentration of the one substance.
  • the relative concentration of the one substance is, for example, a relative area value CE.
  • the relative concentration of the one substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples.
  • the relative concentration of the one substance is, for example, a relative area value LC.
  • the relative concentration of the one substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • measurement values to be acquired in step 1a are, for example, the relative concentrations or absolute concentrations of the two or more substances, and preferably the absolute concentrations of the two or more substances.
  • the method for measuring the substances is CE-MS (preferably CE-FTMS)
  • the relative concentration of each substance is, for example, a relative area value CE.
  • the relative concentration of each substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples.
  • the method for measuring the substances is LC-MS (preferably LC-MS/MS)
  • the relative concentration of each substance is, for example, a relative area value LC.
  • the relative concentration of each substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • a measurement value to be acquired in step 1a is, for example, the combined variable determined from the concentrations of the two or more substances, and preferably the combined variable determined from the concentrations of the two or more substances on the basis of the expression (1).
  • the concentrations to be used in determining the combined variable may be relative concentrations or absolute concentrations, but are preferably absolute concentrations.
  • step 1a a measurement value for the one substance is compared with a reference value for the one substance to acquire a comparison result in step 1b.
  • the reference value for the one substance is also a relative concentration.
  • the latter term "relative concentration” is synonymous with the former term “relative concentration”, and the latter relative concentration is determined in the same manner as for the former relative concentration.
  • the measurement value for the one substance is a relative area value CE
  • the reference value for the one substance is also a relative area value CE.
  • the measurement value for the one substance is a relative area value LC
  • the reference value for the one substance is also a relative area value LC.
  • the reference value for the one substance is also an absolute concentration.
  • absolute concentration is synonymous with the former term “absolute concentration”
  • absolute concentration is determined in the same manner as for the former absolute concentration.
  • step 1a In the case where two or more substances in the specimen derived from the subject are measured in step 1a, measurement values for the two or more substances are compared with reference values for the two or more substances to acquire a comparison result in step 1b.
  • the measurement value for the substance of No. 1 is compared with the reference value for the substance of No. 1
  • the measurement value for the substance of No. 2 is compared with the reference value for the substance of No. 2, thereby acquiring a comparison result in step 1b.
  • the reference values for the two or more substances are also relative concentrations.
  • concentration is synonymous with the former term “relative concentration”, and the latter relative concentrations are determined in the same manner as for the former relative concentrations.
  • the measurement values for the substances are relative area values CE
  • the reference values for the substances are also relative area values CE.
  • the measurement values for the substances are relative area values LC
  • the reference values for the substances are also relative area values LC.
  • the reference values for the two or more substances are also absolute concentrations.
  • absolute concentration is synonymous with the former term “absolute concentration”
  • absolute concentrations are determined in the same manner as for the former absolute concentrations.
  • the reference value for the two or more substances is also a combined variable determined from the concentrations of the two or more substances (preferably, a combined variable determined from the concentrations of the two or more substances on the basis of the expression (1)).
  • combined variable is synonymous with the former term “combined variable”, and the latter combined variable is determined in the same manner as for the former combined variable.
  • Step 1c is a step of evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step 1b.
  • the term “positive” means that the intestinal barrier function in the subject has been reduced, and the term “negative” means that the intestinal barrier function in the subject has not been reduced.
  • the intestinal barrier function to be evaluated may be the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, but is preferably the intestinal barrier function of the small intestine.
  • the intestinal barrier function to be evaluated is the physical barrier function including tight junction structures.
  • the intestinal barrier function in the subject can be evaluated on the basis of whether the comparison result acquired in step 1b satisfies the criterion shown in Table 4. Specifically, for each substance being "high" with respect to the criterion shown in Table 4, if the measurement value acquired in step 1a is higher than the reference value (i.e., the comparison result acquired in step 1b satisfies the criterion shown in Table 4), the case can be evaluated as being positive; if the measurement value acquired in step 1a is equal to or lower than the reference value (i.e., the comparison result acquired in step 1b does not satisfy the criterion shown in Table 4), the case can be evaluated as being negative.
  • the case can be evaluated as being positive; if the measurement value acquired in step 1a is equal to or higher than the reference value (i.e., the comparison result acquired in step 1b does not satisfy the criterion shown in Table 4), the case can be evaluated as being negative.
  • the criterion shown in Table 4 can also be applied to aspects other than Aspect 1.
  • one substance selected from the substance group of the present invention is measured in step 1a, if a comparison result for the one substance satisfies the criterion shown in Table 4, the case can be evaluated as being positive; if a comparison result for the one substance does not satisfy the criterion shown in Table 4, the case can be evaluated as being negative.
  • the reference value for each substance selected from the substance group of the present invention can be set according to a conventional method.
  • the average value of measurement values for the substance in specimens obtained from a negative group (a population with non-reduced intestinal barrier function) consisting of individuals of the same species as the subject (e.g., humans if the subject is a human) can be used as a reference value for the substance.
  • a negative group a population with non-reduced intestinal barrier function
  • this embodiment hereinafter, referred to as "Embodiment 1-1" will be described.
  • the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test).
  • a known testing method for intestinal barrier function e.g., lactulose/mannitol test.
  • the specimens obtained from the negative group are also blood.
  • the specimens obtained from the negative group are also serum.
  • the specimens obtained from the negative group are also plasma.
  • measurement value for each substance in the specimens obtained from the negative group is synonymous with that for the measurement value acquired in step 1a, and the measurement values are determined in the same manner as in step 1a.
  • the reference value for the one substance to be used in step 1b is the average value of the relative concentrations of the one substance in the specimens obtained from the negative group.
  • the latter term "relative concentration” is synonymous with the former term “relative concentration”, and the latter relative concentrations are determined in the same manner as for the former relative concentration.
  • the former relative concentration is a relative area value CE
  • the latter relative concentrations are also relative area values CE.
  • the former relative concentration is a relative area value LC
  • the latter relative concentrations are also relative area values LC.
  • the reference value for the one substance to be used in step 1b is the average value of the absolute concentrations of the one substance in the specimens obtained from the negative group.
  • absolute concentration is synonymous with the former term absolute concentration, and the latter absolute concentrations are determined in the same manner as for the former absolute concentration.
  • the reference value for each substance to be used in step 1b is the average value of the relative concentrations of the substance in the specimens obtained from the negative group.
  • the latter term "relative concentration” is synonymous with the former term “relative concentration”, and the latter relative concentrations are determined in the same manner as for the former relative concentrations.
  • the former relative concentrations are relative area values CE
  • the latter relative concentrations are also relative area values CE.
  • the former relative concentrations are relative area values LC
  • the latter relative concentrations are also relative area values LC.
  • the reference value for each substance to be used in step 1b is the average value of the absolute concentrations of the substance in the specimens obtained from the negative group.
  • intestinal barrier function in the subject on the basis of whether the comparison result acquired in step 1b satisfies the criterion shown in Table 5.
  • a preferred criterion of x times or more, a more preferred criterion of y times or more, and an even more preferred criterion of z times or more in Table 5 if the measurement value acquired in step 1a is x times or more the average value, the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is y times or more the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is z times or more the average value, the case is even more preferably evaluated as being positive.
  • a preferred criterion of x times or less, a more preferred criterion of y times or less, and an even more preferred criterion of z times or less in Table 5 if the measurement value acquired in step 1a is x times or less the average value, the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is y times or less the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is z times or less the average value, the case is even more preferably evaluated as being positive.
  • the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is 0.7 times or less the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is 0.6 times or less the average value, the case is even more preferably evaluated as being positive.
  • the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is 2.0 times or more the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is 2.5 times or more the average value, the case is even more preferably evaluated as being positive.
  • Table 5-1 Table 5 (Criterion) No.
  • Substance name Criterion Preferred criterion More preferred criterion Even more preferred criterion 1 Melamine Low 0.9 times or less 0.7 times or less 0.6 times or less 2 Azelaic acid High 1.5 times or more 2.0 times or more 2.5 times or more 3 Urocanic acid Low 0.9 times or less 0.7 times or less 0.5 times or less 4 Acetanilide Low 0.9 times or less 0.7 times or less 0.5 times or less 5 Paraxanthine Low 0.7 times or less 0.5 times or less 0.3 times or less 6 4-Acetamidobutanoic acid Low 0.9 times or less 0.8 times or less 0.6 times or less 7 Glycerol High 1.1 times or more 1.2 times or more 1.3 times or more 8 N-Acetylputrescine Low 0.9 times or less 0.8 times or less 0.6 times or less 9 N8-Acetylspermidine Low 0.9 times or less 0.8 times or less 0.6 times or less 10 Threonine Low 0.95 times or less 0.9 times or less 0.7 times or less 11
  • Substance name Criterion Preferred criterion More preferred criterion Even more preferred criterion 24 Betonicine Low 0.1 times or less 0.06 times or less 0.01 times or less 25 Adenine High 8 times or more 10 times or more 15 times or more 26 N-Methylnicotinamide High 1.05 times or more 1.1 times or more 1.2 times or more 27 4-Hexyloxyaniline High 1.1 times or more 1.3 times or more 1.6 times or more 28 Caffeine Low 0.8 times or less 0.6 times or less 0.4 times or less 29 Theophylline Low 0.8 times or less 0.6 times or less 0.4 times or less 30 DL- ⁇ -Leucine High 1.05 times or more 1.1 times or more 1.2 times or more 31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide High 10 times or more 30 times or more 40 times or more 32 Guanidineacetic acid Low 0.9 times or less 0.8 times or less 0.6 times or less 33
  • the median of measurement values for the substance in specimens obtained from a negative group (a population with non-reduced intestinal barrier function) consisting of individuals of the same species as the subject (e.g., humans if the subject is a human) can be used as a reference value for the substance.
  • a negative group a population with non-reduced intestinal barrier function
  • this embodiment hereinafter, referred to as "Embodiment 1-2" will be described.
  • Embodiment 1-1 is also applied to Embodiment 1-2.
  • the term “average value” is replaced with the term “median”.
  • a cutoff value obtained from an ROC (Receiver Operating Characteristic) curve for the substance can be used as a reference value for the substance.
  • ROC Receiveiver Operating Characteristic
  • the ROC curve for each substance can be prepared according to a conventional method on the basis of the concentrations of the substance in specimens obtained from a negative group (a population with non-reduced intestinal barrier function) and a positive group (a population with reduced intestinal barrier function) each consisting of individuals of the same species as the subject (e.g., humans if the subject is a human).
  • the ordinate represents sensitivity
  • the abscissa represents (1 - specificity).
  • the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test).
  • the specimens obtained from the negative group and positive group are also blood.
  • the specimens obtained from the negative group and positive group are also serum.
  • the specimens obtained from the negative group and positive group are also plasma.
  • the concentrations of the one substance to be used for preparation of the ROC curve for the one substance are also relative concentrations.
  • the latter term "relative concentration” is synonymous with the former term “relative concentration”, and the latter relative concentrations are determined in the same manner as for the former relative concentrations.
  • the former relative concentration is a relative area value CE
  • the latter relative concentrations are also relative area values CE.
  • the former relative concentration is a relative area value LC
  • the latter relative concentrations are also relative area values LC.
  • the cutoff value to be obtained from the ROC curve for the one substance is also a relative concentration.
  • the concentrations of the one substance to be used for preparation of the ROC curve for the one substance are also absolute concentrations.
  • the latter absolute concentrations are determined in the same manner as for the former absolute concentration.
  • the cutoff value to be obtained from the ROC curve for the one substance is also an absolute concentration.
  • the concentrations of each substance to be used for preparation of the ROC curve for the substance are also relative concentrations.
  • the latter term "relative concentration” is synonymous with the former term “relative concentration”, and the latter relative concentrations are determined in the same manner as for the former relative concentrations.
  • the former relative concentrations are relative area values CE
  • the latter relative concentrations are also relative area values CE.
  • the former relative concentrations are relative area values LC
  • the latter relative concentrations are also relative area values LC.
  • the cutoff value to be obtained from the ROC curve for the substance is also a relative concentration.
  • the concentrations of each substance to be used for preparation of the ROC curve for the substance are also absolute concentrations.
  • the latter absolute concentrations are determined in the same manner as for the former absolute concentrations.
  • the cutoff value to be obtained from the ROC curve for the substance is also an absolute concentration.
  • a cutoff value is such a value that both high sensitivity and high specificity can be achieved when evaluation is carried out with reference to the value.
  • a cutoff value can be appropriately set in consideration of the balance between sensitivity and specificity. For example, a value at which a high positive rate is exhibited in the positive group and a high negative rate is exhibited in the negative group can be set as a cutoff value.
  • a method for setting a cutoff value is well known to those skilled in the art.
  • Known examples therefor include a method in which a point on an ROC curve at which the distance from the upper left corner of the ROC curve is minimized is used as a cutoff value, and a method in which a point on an ROC curve at which the value of (sensitivity + specificity - 1) is maximized is used as a cutoff value.
  • the point on an ROC curve at which the value of (sensitivity + specificity - 1) is maximized is called "Younden index".
  • cutoff values for substances selected from the substance group of the present invention are shown in Table 9.
  • the cutoff value for each substance selected from the substance group of the present invention is preferably a value within the range of a value shown as P1 in Table A or more and a value shown as P3 in Table A or less.
  • the cutoff value for each substance selected from the substance group of the present invention may be a value within the range of a value shown as P1 in Table A or more and a value shown as P2 in Table A or less, or a value within the range of a value shown as P2 in Table A or more and a value shown as P3 in Table A or less.
  • the cutoff value for each substance selected from the substance group of the present invention may be, for example, a value shown as P1 in Table A, or a value shown as P2 in Table A, or a value shown as P3 in Table A. [Table A-1] Table A No.
  • a cutoff value for the two or more substances can be used as a reference value for the two or more substances, wherein the cutoff value is obtained in such a manner that the concentrations of the two or more substances are measured for specimens obtained from a negative group (a population with non-reduced intestinal barrier function) and a positive group (a population with reduced intestinal barrier function) each consisting of individuals of the same species as the subject (e.g., humans if the subject is a human), a combined variable is determined from the concentrations of the two or more substances on the basis of the expression (1), an ROC curve is prepared on the basis of the combined variable determined, and the cutoff value is determined from the ROC curve prepared.
  • this embodiment hereinafter, referred to as "Embodiment 1-4" will be described.
  • Embodiment 1-4 is applied to the case where the measurement value acquired in step 1a is a combined variable determined from the concentrations of two or more substances selected from the substance group of the present invention on the basis of the expression (1).
  • the concentrations to be used for determination of a cutoff value are also relative concentrations.
  • concentration is synonymous with the former term “relative concentration”, and the latter relative concentrations are determined in the same manner as for the former relative concentrations.
  • the former relative concentrations are relative area values CE
  • the latter relative concentrations are also relative area values CE.
  • the former relative concentrations are relative area values LC
  • the latter relative concentrations are also relative area values LC.
  • the concentrations to be used for determination of a cutoff value are also absolute concentrations.
  • absolute concentration is synonymous with the former term “absolute concentration”, and the latter absolute concentrations are determined in the same manner as for the former absolute concentrations.
  • the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test).
  • An ROC curve can be prepared according to a conventional method on the basis of a combined variable obtained from the negative group and positive group.
  • the ordinate represents sensitivity
  • the abscissa represents (1 - specificity).
  • the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test). For cutoff values, the description thereof given for Embodiment 1-3 is applied.
  • the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by CE-FTMS under conditions described in the Examples, specific examples of the coefficients and constant term are as shown in Table 2, and specific examples of the cutoff values are as shown in Table 6.
  • Table 6 “CP_A”, "CP_B”, “CP_C”, and “CP_D” are synonymous with those in Table 2. [Table 6] Table 6 (No. 1 to No.
  • Each of the cutoff values of No. 1 to No. 11 in Table 6 is preferably a value within the range of a value shown as Q1 in Table B or more and a value shown as Q3 in Table B or less.
  • Each of the cutoff values of No. 1 to No. 11 in Table 6 may be a value within the range of a value shown as Q1 in Table B or more and a value shown as Q2 in Table B or less, or a value within the range of a value shown as Q2 in Table B or more and a value shown as Q3 in Table B or less.
  • 11 in Table 6 may be a value shown as Q1 in Table B, or a value shown as Q2 in Table B, or a value shown as Q3 in Table B.
  • Table B No. Q1 Q2 Q3 1 0.3132 0.4474 0.5816 2 -0.1017 -0.0782 -0.0547 3 0.0751 0.1073 0.1395 4 -0.1876 -0.1443 -0.1010 5 0.0718 0.1026 0.1334 6 0.2231 0.3187 0.4143 7 -0.5723 -0.4402 -0.3081 8 0.3342 0.4774 0.6206 9 -0.0638 -0.0491 -0.0344 10 0.0026 0.0037 0.0048 11 0.0568 0.0812 0.1056
  • the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL- ⁇ -leucine), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by LC-MS/MS under conditions described in the Examples, specific examples of the coefficients and constant term are as shown in Table 3, and specific examples of the cutoff values are as shown in Table 7. In Table 7, “LP_A”, “LP_B”, “LP_C”, and "LP_D” are synonymous with those in Table 3. [Table 7] Table 7 (No. 27 to No.
  • Each of the cutoff values of No. 1 to No. 11 in Table 7 is preferably a value within the range of a value shown as R1 in Table C or more and a value shown as R3 in Table C or less.
  • Each of the cutoff values of No. 1 to No. 11 in Table 7 may be a value within the range of a value shown as R1 in Table C or more and a value shown as R2 in Table C or less, or a value within the range of a value shown as R2 in Table C or more and a value shown as R3 in Table C or less.
  • 11 in Table 7 may be a value shown as R1 in Table C, or a value shown as R2 in Table C, or a value shown as R3 in Table C.
  • Table C Table C No. R1 R2 R3 1 0.1587 0.2267 0.2947 2 0.1522 0.2174 0.2826 3 0.0599 0.0855 0.1112 4 -0.2278 -0.1752 -0.1226 5 -0.2643 -0.2033 -0.1423 6 -0.2653 -0.2041 -0.1429 7 -0.0761 -0.0585 -0.0410 8 0.0488 0.0697 0.0906 9 0.1189 0.1699 0.2209 10 -0.0485 -0.0373 -0.0261 11 0.0978 0.1397 0.1816
  • Aspect 2 of the present invention relates to a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the disease or symptom”.
  • the method according to Aspect 2 of the present invention includes the steps of:
  • Aspect 1 is also applied to Aspect 2.
  • step 1a is replaced with “step 2a”
  • step 1b is replaced with “step 2b”
  • step 1c is replaced with “step 2c”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “diagnosing the disease or symptom in the subject”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of diagnosing the disease or symptom in the subject”
  • the phrase “evaluating as being positive” is replaced with the phrase “diagnosing as being positive”
  • the phrase “evaluating as being negative” is replaced with the phrase “diagnosing as being negative”.
  • the term “positive” means that the subject is affected by the disease or symptom, and the term “negative” means that the subject is not affected by the disease or symptom.
  • the disease or symptom to be diagnosed may be a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • Examples of the disease or symptom caused by reduction in intestinal barrier function include enteritis, allergic diseases, psychiatric diseases, non-alcoholic fatty liver diseases, type II diabetes mellitus, metabolic syndrome, and adiposity.
  • Examples of the enteritis include irritable bowel syndrome, ulcerative colitis, and Crohn's disease.
  • Examples of the allergic diseases include food allergies, atopic dermatitis, and asthma.
  • Examples of the psychiatric diseases include anxiety disorder and depression.
  • Examples of the non-alcoholic fatty liver diseases include non-alcoholic fatty liver and non-alcoholic steatohepatitis.
  • Diagnosis is a practice typically performed by a physician.
  • a physician diagnoses whether the subject is affected by the disease or symptom caused by reduction in intestinal barrier function on the basis of the comparison result acquired in step 2b; if the subject is affected by the disease or symptom caused by reduction in intestinal barrier function, the physician may determine whether prevention or treatment of the disease or symptom is needed for the subject, or select a method for preventing or treating the disease or symptom for the subject, or perform prevention or treatment of the disease or symptom for the subject.
  • prevention includes prevention, suppression, and retardation of the onset of the disease or symptom.
  • treatment includes suppression of the progress or exacerbation of the disease or symptom, retardation of the progress or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • the method according to Aspect 2 of the present invention may be a method for assisting diagnosis of a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • the practice of assisting diagnosis of the disease or symptom is a practice of providing the comparison result acquired in step 2b as data useful for diagnosis of the disease or symptom, and this may be a medical practice or a non-medical practice, but is typically a non-medical practice.
  • Diagnosis by a physician can be performed on the basis of the data provided through the practice of assisting diagnosis of the disease or symptom plus additional one or two or more pieces of information. Diagnosis by a physician can involve the experience, techniques, and the like of the physician.
  • Aspect 3 of the present invention relates to a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the disease or symptom”.
  • the method according to Aspect 3 of the present invention includes the steps of:
  • step 2a is replaced with “step 3a”
  • step 2b is replaced with “step 3b”
  • step 2c is replaced with “step 3c”.
  • treatment includes suppression of the progression or exacerbation of a disease or symptom, retardation of the progression or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • Substances or compositions for treating irritable bowel syndrome include high-molecular-weight polymers and serotonin receptor antagonists.
  • Substances or compositions for treating ulcerative colitis or Crohn's disease include 5-aminosalicylate preparations and corticosteroids.
  • Substances or compositions for treating allergic diseases include antihistamines.
  • Substances or compositions for treating anxiety disorder or depression include selective serotonin reuptake inhibitors and noradrenergic antidepressants.
  • Substances or compositions for treating non-alcoholic fatty liver diseases include antioxidants, therapeutics for diabetes mellitus, and therapeutics for dyslipidemia.
  • the substance or composition for treating the disease or symptom may be administered in the form of a food composition containing the substance or composition.
  • a food composition containing the substance or composition.
  • examples of the food composition include yogurt, probiotics, prebiotics, and supplements.
  • the food composition may be, for example, a food for specified health uses, a food with functional claims, or a food for medical use.
  • Examples of the route of administration of the substance or composition for treating the disease or symptom include oral and parenteral (e.g., intranasal, ophthalmic, ear-drop, transdermal, tracheobronchial, intrarectal, urinary, subcutaneous, intramuscular, intravenous) administrations.
  • the substance or composition for treating the disease or symptom may be formulated into a dosage form suitable for the route of administration. Examples of the dosage form include a tablet and an injection.
  • a proper excipient for oral administration e.g., a diluent, a disintegrant, a lubricant, a binder
  • an excipient suitable for parenteral administration e.g., a diluting agent, a solvent
  • the dose and frequency of administration of the substance or composition for treating the disease or symptom can be appropriately adjusted in consideration of the dosage form and the age, body weight, and so on of the subject.
  • the frequency of administration per day may be once, or two or more times.
  • the period of administration may be 1 day or 2 days or more, (e.g., 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more).
  • Aspect 4 of the present invention relates to a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom in a subject.
  • a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the risk of the development”
  • probability of the subject being affected by a disease or symptom caused by reduction in intestinal barrier function may be referred to as "the probability of being affected”.
  • the method according to Aspect 4 includes the steps of:
  • Aspect 1 is applied to Aspect 4.
  • step 1a is replaced with “step 4a”
  • step 1b is replaced with “step 4b”
  • step 1c is replaced with “step 4c”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “evaluating the risk of the development or the probability of being affected”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of evaluating the risk of the development or the probability of being affected”.
  • the term "positive” means that the risk of the development or the probability of being affected is present or high, and the term “negative” means that the risk of the development or the probability of being affected is absent or low.
  • the risk of the development to be evaluated may be a subject's risk of developing a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a subject's risk of developing a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a subject's risk of developing a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • the probability of being affected to be evaluated may be probability of the subject being affected by a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or probability of the subject being affected by a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably probability of the subject being affected by a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • Aspect 5 of the present invention relates to a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the disease or symptom”.
  • the method according to Aspect 5 includes the steps of:
  • Aspect 1 is applied to Aspect 5.
  • step 1a is replaced with “step 5a”
  • step 1b is replaced with “step 5b”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “acquiring data for diagnosing the disease or symptom in the subject”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of diagnosing the disease or symptom in the subject”.
  • the term “positive” means that the subject is affected by the disease or symptom, and the term “negative” means that the subject is not affected by the disease or symptom.
  • the disease or symptom to be evaluated may be a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • a comparison result satisfying the criterion shown in Table 4 can be acquired as data for diagnosing as being positive, and a comparison result not satisfying the criterion shown in Table 4 can be acquired as data for diagnosing as being negative.
  • a comparison result that the measurement value acquired in step 5a is higher than the reference value can be acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is equal to or lower than the reference value can be acquired as data for diagnosing as being negative.
  • a comparison result that the measurement value acquired in step 5a is lower than the reference value can be acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is equal to or higher than the reference value can be acquired as data for diagnosing as being negative.
  • one substance selected from the substance group of the present invention is measured in step 5a, if a comparison result for the one substance satisfies the criterion shown in Table 4, the comparison result can be acquired as data for diagnosing as being negative; if a comparison result for the one substance does not satisfy the criterion shown in Table 4, the comparison result can be acquired as data for diagnosing as being negative.
  • step 5a if a comparison result for at least one substance of the two or more substances satisfies the criterion shown in Table 4, the comparison result can be acquired as data for diagnosing as being positive; if none of comparison results for the two or more substances satisfies the criterion shown in Table 4, the comparison results can be acquired as data for diagnosing as being negative.
  • Embodiment 1-1 of Aspect 1 is applied to Aspect 5
  • a comparison result based on the criterion shown in Table 5 is preferably acquired as data for diagnosis.
  • a comparison result that the measurement value acquired in step 5a is x times or more the average value is preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is y times or more the average value is more preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is z times or more the average value is even more preferably acquired as data for diagnosing as being positive.
  • a comparison result that the measurement value acquired in step 5a is x times or less the average value is preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is y times or less the average value is more preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is z times or less the average value is even more preferably acquired as data for diagnosing as being positive.
  • a comparison result that the measurement value acquired in step 5a is 0.9 times or less the average value is preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is 0.7 times or less the average value is more preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is 0.6 times or less the average value is even more preferably acquired as data for diagnosing as being positive.
  • a comparison result that the measurement value acquired in step 5a is 1.5 times or more the average value is preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is 2.0 times or more the average value is more preferably acquired as data for diagnosing as being positive
  • a comparison result that the measurement value acquired in step 5a is 2.5 times or more the average value is even more preferably acquired as data for diagnosing as being positive.
  • the data acquired in step 5b can be used for diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject, or for assisting diagnosis of the disease or symptom caused by reduction in intestinal barrier function in the subject.
  • Diagnosis is a practice typically performed by a physician.
  • a physician diagnoses whether the subject is affected by the disease or symptom caused by reduction in intestinal barrier function on the basis of the data acquired in step 5b; if the subject is affected by the disease or symptom caused by reduction in intestinal barrier function, the physician may determine whether prevention or treatment of the disease or symptom is needed for the subject, or select a method for preventing or treating the disease or symptom for the subject, or perform prevention or treatment of the disease or symptom for the subject.
  • prevention includes prevention, suppression, and retardation of the onset of the disease or symptom.
  • treatment includes suppression of the progress or exacerbation of the disease or symptom, retardation of the progress or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • the practice of assisting diagnosis of the disease or symptom is a practice of providing the data acquired in step 5b as data for diagnosis of the disease or symptom, and this may be a medical practice or a non-medical practice, but is typically a non-medical practice. Diagnosis by a physician can be performed on the basis of the data provided through the practice of assisting diagnosis of the disease or symptom plus additional one or two or more data.
  • Aspect 6 of the present invention relates to a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function.
  • a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the preventing or ameliorating effect on the disease or symptom”.
  • the method according to Aspect 6 includes the steps of:
  • Aspect 1 is applied to Aspect 6.
  • step 1a is replaced with “step 6b”
  • step 1b is replaced with “step 6c”
  • step 1c is replaced with “step 6d”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom”.
  • the term “positive” means that the candidate substance or candidate composition has the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom
  • the term “negative” means that the candidate substance or candidate composition does not have the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • prevention includes prevention, suppression, and retardation of the onset of the disease or symptom.
  • amelioration includes suppression of the progression or exacerbation of the disease or symptom, retardation of the progression or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • the improving effect on intestinal barrier function to be evaluated may be an improving effect on the intestinal barrier function of the small intestine, or an improving effect on the intestinal barrier function of the large intestine, but is preferably an improving effect on the intestinal barrier function of the small intestine.
  • the preventing or ameliorating effect on the disease or symptom to be evaluated may be a preventing or ameliorating effect on a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a preventing or ameliorating effect on a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a preventing or ameliorating effect on a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • the method according to Aspect 6 allows screening to obtain a candidate substance or candidate composition evaluated to have the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom as a substance or composition having the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • Step 6a is a step of administering a candidate substance or candidate composition to a subject having reduced intestinal barrier function.
  • the reduced intestinal barrier function in the subject can be confirmed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test), or by the method according to Aspect 1.
  • a known testing method for intestinal barrier function e.g., lactulose/mannitol test
  • the method according to Aspect 1 e.g., lactulose/mannitol test
  • the method according to Aspect 6 may include a step of evaluating intestinal barrier function in a subject to confirm reduced intestinal barrier function in the subject by the method according to Aspect 1 before step 6a.
  • the biomarker to be measured in step 1a in Aspect 1 may be the same as or different from the biomarker to be measured in step 6b in terms of type, but they are preferably the same for enhanced accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • the method according to Aspect 6 may include a step of selecting a subject having reduced intestinal barrier function by the method according to Aspect 1 before step 6a.
  • the biomarker to be measured in step 1a in Aspect 1 may be the same as or different from the biomarker to be measured in step 6b in terms of type, but they are preferably the same for enhanced accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • the subject to which the candidate substance or candidate composition is to be administered is not limited as long as the intestinal barrier function has been reduced, and may be a human having reduced intestinal barrier function or a model animal (except humans) having reduced intestinal barrier function, but is preferably a model animal (except humans) having reduced intestinal barrier function.
  • Such model animals include vertebrates including mammals, reptiles, birds, amphibians, and fish, mammals and birds are preferred, and mammals are more preferred.
  • the mammals include primates (e.g., gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), domestic animals (e.g., cattle, pigs, sheep, goats, horses), and pet animals (e.g., dogs, cats).
  • the birds include poultry (e.g., chickens, wild ducks, ducks).
  • the candidate substance or candidate composition to be administered to the subject having reduced intestinal barrier function is not limited, and can be selected from, for example, high-molecular-weight compounds, low-molecular-weight compounds, cell cultures, cell extracts, antibodies, proteins, peptides, nucleic acids, carbohydrates, inorganic salts, metal complexes, microorganisms (bacteria (probiotics), yeasts, etc.), dietary fibers, resistant carbohydrates, resistant proteins, fermented products (including fermented foods), prebiotics, and any combination of two or more of these.
  • Examples of the route of administration of the candidate substance or candidate composition to the subject include oral and parenteral (e.g., intranasal, ophthalmic, ear-drop, transdermal, tracheobronchial, intrarectal, urinary, subcutaneous, intramuscular, intravenous) administrations.
  • the candidate substance or candidate composition may be formulated into a dosage form suitable for the route of administration. Examples of the dosage form include a tablet and an injection.
  • a proper excipient for oral administration e.g., a diluent, a disintegrant, a lubricant, a binder
  • an excipient suitable for parenteral administration e.g., a diluting agent, a solvent
  • the dose and frequency of administration of the candidate substance or candidate composition can be appropriately adjusted in consideration of the dosage form and the age, body weight, and so on of the subject.
  • the frequency of administration per day may be once, or two or more times.
  • the period of administration may be 1 day or 2 days or more (e.g., 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more).
  • Step 6b is a step of measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker.
  • Step 6b can be performed in the same manner as step 1a in Aspect 1, except that a specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject is used.
  • the specimen to be used in step 6b is obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject.
  • the specimen may be obtained from the subject at multiple time points after administration of the candidate substance or candidate composition.
  • the time point to obtain the specimen can be appropriately adjusted in consideration of the dosage form and the age, body weight, and so on of the subject.
  • the time point to obtain the specimen is, for example, any time point between 0.5 and 24 hours after administration, or any time point between 0.5 and 12 hours after administration, or any time point between 1 and 10 hours after administration.
  • the meaning of "after administration” may be "after one administration" or "after two or more continuous administrations".
  • the meaning of "after two or more continuous administrations” may be, for example, “after the final administration of continuous administrations for 4 weeks or more", or “after the final administration of continuous administrations for 1 day to 4 weeks", or “after the final administration of continuous administrations for 3 days to 3 weeks", or "after the final administration of continuous administrations for 1 week to 2 weeks".
  • Step 6c is a step of comparing the measurement value acquired in step 6b with a reference value to acquire a comparison result.
  • Step 6c can be performed in the same manner as step 1b in Aspect 1, except that the measurement value acquired in step 6b is used.
  • Step 6d is a step of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 6c.
  • Step 6d can be performed in the same manner as step 1c in Aspect 1, except that the candidate substance or candidate composition is evaluated for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • the candidate substance or candidate composition can be evaluated for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of whether the comparison result acquired in step 6c satisfies the criterion shown in Table 4.
  • the case can be evaluated as being negative; if the measurement value acquired in step 6b is equal to or lower than the reference value (i.e., the comparison result acquired in step 6c does not satisfy the criterion shown in Table 4), the case can be evaluated as being positive.
  • the case can be evaluated as being negative; if the measurement value acquired in step 6b is equal to or higher than the reference value (i.e., the comparison result acquired in step 6c does not satisfy the criterion shown in Table 4), the case can be evaluated as being positive.
  • one substance selected from the substance group of the present invention is measured in step 6b, if a comparison result for the one substance satisfy the criterion shown in Table 4, the case can be evaluated as being negative; if a comparison result for the one substance does not satisfy the criterion shown in Table 4, the case can be evaluated as being positive.
  • two or more substances selected from the substance group of the present invention are measured in step 6b, if all comparison results for the two or more substances satisfy the criteria shown in Table 4, the case can be evaluated as being negative; if a comparison result for at least one substance of the two or more substances does not satisfy the criterion shown in Table 4, the case can be evaluated as being positive.
  • two or more (e.g., two, three, or four or more) substances selected from the substance group of the present invention can be measured in step 6b to achieve enhanced accuracy of evaluating a candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • Aspect 7 of the present invention relates to a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function.
  • a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the preventing or ameliorating effect on the disease or symptom”.
  • the method according to Aspect 7 includes the steps of:
  • step 6a is replaced with “step 7a”
  • step 6b is replaced with “step 7b”
  • step 6c is replaced with “step 7c”
  • step 6d is replaced with "step 7d”.
  • Aspect 8 of the present invention relates to a biomarker in a specimen derived from a subject for use in evaluating intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 9 of the present invention relates to a biomarker in a specimen derived from a subject for use in diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 10 of the present invention relates to a biomarker in a specimen derived from a subject for use in evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 11 of the present invention relates to a biomarker in a specimen derived from a subject for use in acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 12 of the present invention relates to a biomarker in a specimen for use in screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 13 of the present invention relates to a biomarker in a specimen for use in evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 14 of the present invention relates to use of a biomarker in a specimen derived from a subject for evaluating intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 15 of the present invention relates to use of a biomarker in a specimen derived from a subject for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 16 of the present invention relates to use of a biomarker in a specimen derived from a subject for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 17 of the present invention relates to use of a biomarker in a specimen derived from a subject for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 18 of the present invention relates to use of a biomarker in a specimen for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 19 of the present invention relates to use of a biomarker in a specimen for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 20 of the present invention relates to a kit for evaluating intestinal barrier function in a subject, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 1 is applied to Aspect 20.
  • the kit may include one reagent, or two or more reagents.
  • a reagent of any type that fits with the type of method for measuring a target substance can be appropriately selected.
  • the method for measuring a target substance include liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), gas chromatography-mass spectrometry (GC-MS), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), and CE-MS or LC-MS is preferred among these.
  • CE-MS is suitable for measurement of water-soluble substances
  • LC-MS is suitable for measurement of liposoluble substances.
  • CE-FTMS capillary electrophoresis-Fourier transform mass spectrometry
  • Particularly preferred among different types of LC-MS is liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • the kit includes one or more reagents for measuring the concentration of a target substance by CE-MS.
  • concentration may be a relative concentration or an absolute concentration, but is preferably an absolute concentration.
  • the relative concentration is, for example, a relative area value CE.
  • the reagent for measuring the concentration of a target substance by CE-MS include authentic samples of one or more substances selected from the substance group of the present invention.
  • the kit include one or more reagents for measuring the concentration of a target substance by LC-MS.
  • concentration may be a relative concentration or an absolute concentration, but is preferably an absolute concentration.
  • the relative concentration is, for example, a relative area value LC.
  • the reagent for measuring the concentration of a target substance by LC-MS include authentic samples of one or more substances selected from the substance group of the present invention.
  • Aspect 21 of the present invention relates to a kit for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 22 of the present invention relates to a kit for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 23 of the present invention relates to a kit for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 24 of the present invention relates to a kit for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function
  • the kit includes one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 25 of the present invention relates to a kit for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, wherein the kit includes one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Aspect 26 of the present invention relates to a method for producing a food product or pharmaceutical composition, the method including the step of blending a substance or composition obtained through screening by the method according to Aspect 6 with one or two or more components to constitute the food product or pharmaceutical composition.
  • the one or two or more components to be blended with the substance or composition obtained through screening by the method according to Aspect 6 may be any ones acceptable as components for a food product or pharmaceutical composition without limitation, and appropriate ones can be selected to fit with the type of food product or pharmaceutical composition to be produced.
  • the method according to Aspect 26 may include a step of screening for a substance or composition by the method according to Aspect 6.
  • Aspect 27 of the present invention relates to a computer program for allowing a computer to execute a method for evaluating intestinal barrier function in a subject.
  • the method that a computer is allowed to execute by the computer program according to Aspect 27 includes the steps of:
  • the computer program according to Aspect 27 allows a computer to execute a method for evaluating intestinal barrier function in a subject when the computer program is executed by the computer.
  • the computer program according to Aspect 27 includes an instruction to allow a computer to execute a method for evaluating intestinal barrier function in a subject.
  • Figure 1 is a schematic diagram of an apparatus to be used in the present embodiment
  • Figure 2 is a block diagram illustrating the hardware configuration of the apparatus of the present embodiment
  • Figures 3 to 5 are each a flowchart illustrating a procedure with the apparatus of the present embodiment.
  • an apparatus 10 includes a computer system 30.
  • the apparatus 10 may include a measurement device 20 connected to the computer system 30.
  • the computer system 30 may be a system provided separately from the measurement device 20, or a system including the measurement device 20 therein.
  • the apparatus 10 may be an apparatus in which the measurement device 20 and the computer system 30 are integrally configured.
  • the measurement device 20 executes measurement of a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker (e.g., an absolute concentration, a relative concentration, or a combined variable) or a measurement value necessary for calculating a measurement value for the biomarker (e.g., a measurement value necessary for calculating a relative concentration or a combined variable), and sends the acquired value to the computer system 30.
  • a measurement value for the biomarker e.g., an absolute concentration, a relative concentration, or a combined variable
  • a measurement value necessary for calculating a measurement value for the biomarker e.g., a measurement value necessary for calculating a relative concentration or a combined variable
  • the measurement device 20 is an automated measurement device that executes measurement of the biomarker by CE-MS (in particular, CE-FTMS) or LC-MS (in particular, LC-MS/MS) to acquire a measurement value for the biomarker (e.g., a relative area value CE, a relative area value LC, or a combined variable) or a measurement value necessary for calculating a measurement value for the biomarker (e.g., a measurement value necessary for calculating a relative area value CE, a relative area value LC, or a combined variable).
  • a measurement value for the biomarker e.g., a relative area value CE, a relative area value LC, or a combined variable
  • a measurement value necessary for calculating a measurement value for the biomarker e.g., a measurement value necessary for calculating a relative area value CE, a relative area value LC, or a combined variable.
  • the measurement device 20 is needed to be able to execute measurement of the biomarker in a measurement sample (e.g., a measurement sample to be used for CE-MS (in particular, CE-FTMS) or LC-MS (in particular, LC-MS/MS)) and the subsequent process.
  • a measurement sample e.g., a measurement sample to be used for CE-MS (in particular, CE-FTMS) or LC-MS (in particular, LC-MS/MS)
  • Processes prior to measurement of the biomarker in a measurement sample such as collection of blood, serum, or plasma derived from the subject (i.e., collection of blood, serum, or plasma to be used as a specimen from blood obtained from the subject), preparation of a measurement sample from blood, serum, or plasma derived from the subject, and setting of a measurement sample to the measurement device 20 may be executed by a human or by the measurement device 20.
  • processes prior to measurement of the biomarker in a measurement sample are executed by a human.
  • the computer system 30 includes: a computer main body 300; an input section 301; and a display section 302 that displays specimen information, evaluation results, and so on.
  • a processor of the computer system 30 executes the computer program, installed in a hard disk 313, for evaluating intestinal barrier function in the subject.
  • the computer main body 300 includes: a CPU (Central Processing Unit) 310; a ROM (Read Only Memory) 311; a RAM (Random Access Memory) 312; a hard disk 313; an input/output interface 314; a reader 315; a communication interface 316; and an image output interface 317.
  • the CPU 310, ROM 311, RAM 312, hard disk 313, input/output interface 314, reader 315, communication interface 316, and image output interface 317 are connected together via buses 318 in a manner that allows data communication.
  • the measurement device 20 is communicatively connected to the computer system 30 via the communication interface 316.
  • the CPU 310 is capable of executing a program stored in the ROM 311 or hard disk 313 or a program loaded in the RAM 312. Through execution of a program by the CPU 310, the apparatus 10 functions as an apparatus for evaluating intestinal barrier function in the subject.
  • the ROM 311 is configured with a mask ROM, a PROM, an EPROM, an EEPROM, or the like.
  • a computer program to be executed by the CPU 310 and data to be used for execution of the computer program are stored in the ROM 311.
  • the RAM 312 is configured with an SRAM, a DRAM, or the like.
  • the RAM 312 is used for reading programs stored in the ROM 311 and hard disk 313.
  • the RAM 312 is used as a working area for the CPU 310 when a program is executed.
  • an operating system to be executed by the CPU 310 computer programs including application programs, and data to be used for execution of the computer programs have been installed.
  • the reader 315 is configured with a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a memory stick reader, a solid state drive, or the like.
  • the reader 315 is capable of reading programs or data stored in a transportable storage medium 400.
  • the input/output interface 314 is configured with a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE 1284, and an analog interface consisting of a D/A converter, an A/D converter, or the like.
  • a serial interface such as USB, IEEE 1394, and RS-232C
  • a parallel interface such as SCSI, IDE, and IEEE 1284
  • an analog interface consisting of a D/A converter, an A/D converter, or the like.
  • the input section 301 including a keyboard and a mouse is connected. An operator can input various instructions to the computer main body 300 via the input section 301.
  • the communication interface 316 is, for example, an Ethernet (R) interface.
  • the computer main body 300 is capable of executing sending of print data, for example, to a printer, a computer of the subject or a service provider, or a smartphone terminal of the subject through the communication interface 316.
  • the image output interface 317 is connected to the display section 302 configured with an LCD, a CRT, or the like. With this configuration, the display section 302 is capable of outputting video signals corresponding to image data given by the CPU 310.
  • the display section 302 displays an image (picture) according to inputted video signals.
  • the display section 302 may display raw data themselves, or an illustration, a drawing, or the like made by converting raw data for easy understanding by the subject.
  • step S101 the CPU 310 acquires a measurement value for a biomarker in a specimen derived from the subject.
  • the description thereof given for Aspect 1 is applied.
  • the measurement device 20 executes measurement of a biomarker in a specimen derived from the subject, acquires a measurement value for the biomarker (e.g., an absolute concentration, a relative concentration, or a combined variable), and sends the acquired value to the computer system 30.
  • a measurement value for the biomarker e.g., an absolute concentration, a relative concentration, or a combined variable
  • the description thereof given for Aspect 1 is applied.
  • the CPU 310 stores the measurement value for the biomarker, the measurement value sent from the measurement device 20, in the hard disk 313.
  • the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • the measurement device 20 executes measurement of a biomarker in a specimen derived from the subject, acquires a measurement value necessary for calculating a measurement value for the biomarker (e.g., a measurement value necessary for calculating a relative concentration or a combined variable), and sends the acquired value to the computer system 30.
  • a measurement value necessary for calculating a measurement value for the biomarker e.g., a measurement value necessary for calculating a relative concentration or a combined variable
  • the description thereof given for Aspect 1 is applied.
  • the CPU 310 calculates a measurement value for the biomarker (e.g., a relative concentration or a combined variable), and stores the measurement value for the biomarker in the hard disk 313.
  • the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • an operator inputs a measurement value for a biomarker (e.g., an absolute concentration, a relative concentration, or a combined variable), the measurement value acquired in advance through measurement of the biomarker in a specimen derived from the subject, via the input section 301.
  • a measurement value for a biomarker e.g., an absolute concentration, a relative concentration, or a combined variable
  • the measurement value acquired in advance through measurement of the biomarker in a specimen derived from the subject via the input section 301.
  • the measurement of the biomarker, and the measurement value for the biomarker the description thereof given for Aspect 1 is applied.
  • the CPU 310 stores the inputted measurement value for the biomarker in the hard disk 313.
  • the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • an operator inputs a measurement value necessary for calculating a measurement value for a biomarker (e.g., a measurement value necessary for calculating a relative concentration or a combined variable), the measurement value acquired in advance through measurement of the biomarker in a specimen derived from the subject, via the input section 301.
  • a measurement value necessary for calculating a measurement value for a biomarker e.g., a measurement value necessary for calculating a relative concentration or a combined variable
  • the measurement value acquired in advance through measurement of the biomarker in a specimen derived from the subject via the input section 301.
  • the CPU 310 calculates a measurement value for the biomarker (e.g., a relative concentration or a combined variable), and stores the measurement value for the biomarker in the hard disk 313.
  • the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • step S102 the CPU 310 compares the measurement value for the biomarker with a reference value stored in the hard disk 313 to acquire a comparison result.
  • the reference value the comparison, and the comparison result, the description thereof given for Aspect 1 is applied.
  • the CPU 310 stores the acquired comparison result in the hard disk 313.
  • the CPU 310 may output the acquired comparison result to allow the display section 302 to display the comparison result, or allow a printer to print the comparison result, or transfer the comparison result to another computer or an application (such as a smartphone).
  • the CPU 310 may allow the display section 302 to display the measurement value for the biomarker, the reference value, and others as reference information, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone). Together with the comparison result, the CPU 310 may allow the display section 302 to display advice on life, foods recommended to ingest (including functional foods), and others for improved intestinal barrier function, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone).
  • an index for evaluating intestinal barrier function in the subject can be provided, for example, to a physician, a nurse, a caregiver, a tester, a testing business operator, a service provider, or the subject himself/herself.
  • the CPU 310 evaluates intestinal barrier function in the subject on the basis of the comparison result acquired in step S102. For the evaluation, the description thereof given for Aspect 1 is applied.
  • the CPU 310 stores the evaluation result in the hard disk 313.
  • the CPU 310 may output the evaluation result to allow the display section 302 to display the evaluation result, or allow a printer to print the evaluation result, or transfer the evaluation result to another computer or an application (such as a smartphone).
  • the CPU 310 may allow the display section 302 to display the measurement value for the biomarker, the reference value, and others as reference information, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone).
  • the CPU 310 may allow the display section 302 to display advice on life, foods recommended to ingest (including functional foods), and others for improved intestinal barrier function, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone).
  • an index for evaluating intestinal barrier function in the subject can be provided, for example, to a physician, a nurse, a caregiver, a tester, a testing business operator, a service provider, or the subject himself/herself.
  • the CPU 310 evaluates intestinal barrier function in the subject on the basis of whether the comparison result acquired in step S102 satisfies the criterion shown in Table 4. Specifically, for a substance being "high" with respect to the criterion shown in Table 4, as shown in Figure 4 , the CPU 310 determines whether the measurement value acquired in step 101 is higher than the reference value (i.e., whether the comparison result satisfies the criterion shown in Table 4) (step S103a); if the measurement value acquired in step 101 is higher than the reference value (i.e., the comparison result satisfies the criterion shown in Table 4), the case is evaluated as being positive (step S103b); if the measurement value acquired in step 101 is equal to or lower than the reference value (i.e., the comparison result does not satisfy the criterion shown in Table 4), the case is evaluated as being negative (step S103c).
  • the CPU 310 determines whether the measurement value acquired in step 101 is lower than the reference value (i.e., whether the comparison result satisfies the criterion shown in Table 4) (step S103d); if the measurement value acquired in step 101 is lower than the reference value (i.e., the comparison result satisfies the criterion shown in Table 4), the case is evaluated as being positive (step S103e); if the measurement value acquired in step 101 is equal to or higher than the reference value (i.e., the comparison result does not satisfy the criterion shown in Table 4), the case is evaluated as being negative (step S103f).
  • Aspect 28 of the present invention relates to a computer program for allowing a computer to execute a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the disease or symptom”.
  • the method that a computer is allowed to execute by the computer program according to Aspect 28 includes the steps of:
  • step S101 is replaced with “step S201”
  • step S102 is replaced with “step S202”
  • step S103 is replaced with “step S203”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “diagnosing the disease or symptom in the subject”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of diagnosing the disease or symptom in the subject”
  • the phrase “evaluating as being positive” is replaced with the phrase “diagnosing as being positive”
  • phrase “evaluating as being negative” is replaced with the phrase “diagnosing as being negative”.
  • Aspect 29 of the present invention relates to a computer program for allowing a computer to execute a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom.
  • a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function may be refers to as “the risk of the development”
  • probability of the subject being affected by a disease or symptom caused by reduction in intestinal barrier function may be referred to as "the probability of being affected”.
  • the method that a computer is allowed to execute by the computer program according to Aspect 29 includes the steps of:
  • step S101 is replaced with “step S301”
  • step S102 is replaced with “step S302”
  • step S103 is replaced with “step S303”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “evaluating the risk of the development or the probability of being affected”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of evaluating the risk of the development or the probability of being affected”.
  • Aspect 30 of the present invention relates to a computer program for allowing a computer to execute a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the disease or symptom”.
  • the method that a computer is allowed to execute by the computer program according to Aspect 30 includes the steps of:
  • step S101 is replaced with “step S401”
  • step S102 is replaced with “step S402”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “acquiring data for diagnosing the disease or symptom in the subject”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of diagnosing the disease or symptom in the subject”.
  • Aspect 31 of the present invention relates to a computer program for allowing a computer to execute a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function.
  • a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the preventing or ameliorating effect on the disease or symptom”.
  • the method that a computer is allowed to execute by the computer program according to Aspect 31 includes the steps of:
  • step S101 is replaced with “step S501”
  • step S102 is replaced with “step S502”
  • step S103 is replaced with “step S503”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom”.
  • Aspect 32 of the present invention relates to a computer program for allowing a computer to execute a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function.
  • a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function may be referred to as “the preventing or ameliorating effect on the disease or symptom”.
  • the method that a computer is allowed to execute by the computer program according to Aspect 32 includes the steps of:
  • step S101 is replaced with “step S601”
  • step S102 is replaced with “step S602”
  • step S103 is replaced with “step S603”
  • the phrase “evaluating intestinal barrier function in the subject” is replaced with the phrase “evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom”
  • the phrase “accuracy of evaluating intestinal barrier function in the subject” is replaced with the phrase “accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom”.
  • Aspect 33 of the present invention relates to a computer-readable storage medium storing the computer program according to any one of Aspects 27 to 32.
  • Examples of the computer-readable storage medium storing the program include a non-temporary storage medium such as a ROM, a floppy disk (R), a hard disk, an optical disk, a magneto-optical disk, a CD-ROM, magnetic tape, and a nonvolatile memory card.
  • a non-temporary storage medium such as a ROM, a floppy disk (R), a hard disk, an optical disk, a magneto-optical disk, a CD-ROM, magnetic tape, and a nonvolatile memory card.
  • Lactulose/mannitol test a known testing method for intestinal barrier function, was carried out for 108 test subjects.
  • the lactulose/mannitol test was carried out as follows. Each test subject was instructed to finish his or her meal by 9 p.m. on the day before testing, and allowed to drink water before bedtime. On the day of testing, each test subject, without being allowed to eat or drink water, was subjected to sugar tolerance test. Each test subject was instructed to ingest a sugar solution (prepared by dissolving 10 g of lactulose, 5 g of mannitol, and 20 g of sucrose in 100 mL of water).
  • each test subject was instructed to ingest 150 mL or more of water.
  • the whole of urine excreted during the period of 0 to 6 hours after ingestion of water was collected, and the amounts of lactulose and mannitol in the urine were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • the LC-MS/MS was performed as follows.
  • Each measurement sample to be used for the LC-MS/MS was prepared from urine as follows. To 50 ⁇ L of urine, 400 ⁇ L of cooled acetonitrile was added, and the resultant was sufficiently stirred and left to stand on ice for 1 hour. The resulting mixture was further stirred, 800 ⁇ L of 75% by mass acetonitrile was added, the mixture was sufficiently stirred and filtered through a Millex FH 0.45- ⁇ m filter, and the resultant was used as a measurement sample. No internal standard was used.
  • Excretion rate excretion ( ⁇ mol/mL) ⁇ molecular weight ( ⁇ g/ ⁇ mol) ⁇ urine volume (mL)/amount of ingested sugar ( ⁇ g) ⁇ 100.
  • test subjects with lactulose/mannitol ratios of 0.025 or more were included in a positive group, and twenty-seven test subjects with lactulose/mannitol ratios of 0.015 or less were included in a negative group.
  • Plasma and serum were obtained from the negative group and the positive group, and used in the following comparative example and example.
  • Serum was obtained as follows. On the day of the lactulose/mannitol test, 20 mL of blood was collected from each test subject, and serum was separated from part of the blood and stored in a deep freezer (-80°C) until use.
  • Plasma was obtained as follows. Prior to the sugar tolerance test conducted on the day of test, blood was collected. A portion of the blood was separated into serum and plasma, which were stored in a deep freezer (-80°C) until use.
  • serum zonulin concentration was measured by ELISA.
  • the ELISA was performed with a Zonuline ELISA kit (Immundiagnostik AG, Germany) in accordance with a protocol from the manufacturer.
  • ROC Receiveiver Operating Characteristic
  • AUC area under the curve
  • cutoff value were determined from the ROC curve prepared.
  • the ROC curve, AUC, and cutoff value were determined by using the statistical analysis software "Excel Statistics" (manufactured by Social Survey Research Information Co., Ltd.).
  • the ROC curve is a curve drawn by plotting the serum zonulin concentrations from the negative group and the positive group with an ordinate representing sensitivity and an abscissa representing (1 - specificity).
  • the AUC is the area under the ROC curve.
  • a point on the ROC curve at which the distance from the upper left corner of the ROC curve was minimized was employed as the cutoff value. Table 8 shows the results.
  • metabolomic analysis was performed to comprehensively measure metabolites in the plasma.
  • the metabolomic analysis was performed by capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • Each measurement sample to be used for CE-FTMS was prepared from plasma as follows. To 50 ⁇ L of plasma, 200 ⁇ L of an ethanol solution containing an internal standard (2 ⁇ M) was added, and the resultant was vortexed. To the resultant, 150 ⁇ L of Milli-Q water was added, and the mixture was stirred. A 300 ⁇ L portion of the mixture was transferred to an ultrafiltration tube, centrifuged (4°C, 9100 ⁇ g, 120 minutes), and subjected to ultrafiltration. The filtrate was dried and reconstituted in 50 ⁇ L of Milli-Q water for use as a measurement sample.
  • the internal standard used was an internal standard (H3304-1002; HMT) from Human Metabolome Technologies, Inc. (HMT).
  • CE-FTMS Measurement by CE-FTMS and the subsequent data analysis were performed by using an Agilent CE system (Agilent Technologies). Capillary electrophoresis was performed by using a Fused silica capillary. The conditions for the measurement by CE-FTMS were as follows.
  • Each measurement sample to be used for LC-MS/MS was prepared from plasma as follows. Per 50 ⁇ L of plasma, 200 ⁇ L of cooled methanol containing 0.15% by mass formic acid was added, and the resultant was sufficiently stirred and left to stand on ice for 1 hour.
  • the resulting mixture was centrifuged (12000 rpm, 10 min, 4°C), and a 200- ⁇ L portion was taken from the supernatant and brown with nitrogen gas for exsiccation, and the resultant was dissolved in 200 ⁇ L of 10% by mass methanol (containing 0.1% by mass formic acid) for measurement with an octadecylsilyl (ODS) column, or dissolved in 200 ⁇ L of 50% by mass acetonitrile for measurement with a hydrophilic interaction liquid chromatography (HILIC) column.
  • ODS octadecylsilyl
  • HILIC hydrophilic interaction liquid chromatography
  • HILIC column For measurement with a HILIC column, a product obtained by filtering through a Millex FH 0.45- ⁇ m filter was used as a measurement sample. No internal standard was used. In order to comprehensively analyze metabolites, two columns (ODS column and HILIC column) were used to detect divers substances.
  • ODS column is more suitable for measurement of hydrophobic substances than the HILIC column
  • HILIC column is more suitable for measurement of hydrophilic substances than the ODS column.
  • peaks derived from different substances were specified from peaks detected in CE-FTMS with reference to the m/Z values for the substances, and the peak areas for the substances were determined.
  • the m/Z values for the substances are shown in Table 9.
  • S/N signal/noise
  • Plasma obtained from the negative group (27 individuals) (measurement samples No. 1 to No. 27) and plasma obtained from the positive group (27 individuals) (measurement samples No. 28 to No. 54) were sequentially subjected to measurement by LC-MS/MS. At that time, the relative area value of each substance in each plasma was calculated, with correction of the peak area value for the substance in the measurement sample on the basis of a regression curve prepared from the peak area values for the substances in a mixture (pooled sample) of plasma obtained from the negative group (27 individuals) (measurement samples No. 1 to No. 27) and plasma obtained from the positive group (27 individuals) (measurement samples No. 28 to No. 54) by using the Normalize Area node of the small molecule structure identification software Compound Discoverer 3.1 (Thermo Fisher Scientific).
  • measurement for the pooled sample was performed every 15 samples, and the subsequent data analysis was then performed to determine the peak area for each substance in the pooled sample in the same manner as described above (i.e., the sequence "measurement for measurement samples of No. 1 to No. 15 -> measurement for pooled sample -> measurement for measurement samples of No. 16 to No. 30 -> measurement for pooled sample " was continued until completion of measurement for the sample of No. 54, and the subsequent data analysis was then performed to determine the peak area for each substance in the pooled sample in the same manner as described above).
  • the relative area value of each substance in each plasma was calculated with correction of the peak area value for the substance in the measurement sample in the same manner as described above.
  • the correction with use of the pooled sample was carried out for the purpose of correcting errors, for example, caused by the influence of the temporal attenuation of the peak area values for the substances.
  • ROC Receiveiver Operating Characteristic
  • Table 9 shows the results. Values of AUC closer to 1 indicate higher accuracy of evaluating intestinal barrier function. Cases with a value of AUC of 0.6 or less can be classified as low evaluation accuracy, cases with a value of AUC of more than 0.6 and 0.7 or less as slightly high evaluation accuracy, cases with a value of AUC of more than 0.7 and 0.8 or less as high evaluation accuracy, and cases with a value of AUC of more than 0.8 as very high evaluation accuracy.
  • Table 9 shows results only for substances with AUC of 0.65 or more (the substances of No. 1 to No. 42 in the substance group of the present invention).
  • the positive group average value, the negative group average value, and the ratio of the positive group average value to the negative group average value were determined by using the relative area values for the substance in the plasma.
  • Table 10 shows the results. Table 10 also shows tendencies in the positive group. The entry "increase” indicates the presence of a tendency to increase in the positive group, and the entry “decrease” indicates the presence of a tendency to decrease in the positive group.
  • Substance name Measurement method m/z AUC Cutoff value 1 Melamine CE-FTMS 127.073 0.7791 2.864E-04 2 Azelaic acid CE-FTMS 187.097 0.7366 1.070E-04 3 Urocanic acid CE-FTMS 139.050 0.7188 4.142E-04 4 Acetanilide CE-FTMS 136.076 0.7133 2.701E-04 5 Paraxanthine CE-FTMS 181.072 0.7078 1.032E-02 6 4-Acetamidobutanoic acid CE-FTMS 144.066 0.7078 5.627E-05 7 Glycerol CE-FTMS 93.055 0.7064 8.691E-02 8 N-Acetylputrescine CE-FTMS 131.118 0.7010 1.768E-04 9 N8-Acetylspermidine CE-FTMS 188.176 0.6996 7.304E-05 10 Threonine CE-FTMS 120.066 0.6968 1.428E-01 11 Aminophylline CE-FT
  • a combined variable was determined by using the relative area values obtained in (1) above with an expression shown below, an ROC curve was prepared on the basis of the combined variable determined, and AUC and a cutoff value were determined from the ROC curve prepared. Table 11 shows the results.
  • the coefficients and constant term were determined through multinomial logistic regression analysis. The coefficients and constant term are as shown in Table 2.
  • CP_A refers to melamine
  • CP_B refers to azelaic acid
  • CP_C refers to urocanic acid
  • CP_D refers to acetanilide.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

An object of the present invention is to provide a method for evaluating intestinal barrier function in a subject, and in order to achieve the object, the present invention provides a method for evaluating intestinal barrier function in a subject, the method including the steps of: (1a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker; (1b) comparing the measurement value acquired in step 1a with a reference value to acquire a comparison result; and (1c) evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step 1b, wherein the specimen is blood, serum, or plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.

Description

    TECHNICAL FIELD
  • The present invention relates to a biomarker and use thereof.
  • Specifically, the present invention relates to a method for evaluating intestinal barrier function in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • The present invention also relates to a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the diagnosis, use of a biomarker for the diagnosis, a kit for the diagnosis, and a computer program for allowing a computer to execute the method.
  • The present invention also relates to a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • The present invention also relates to a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • The present invention also relates to a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the acquisition, use of a biomarker for the acquisition, a kit for the acquisition, and a computer program for allowing a computer to execute the method.
  • The present invention also relates to a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the screening, use of a biomarker for the screening, a kit for the screening, and a computer program for allowing a computer to execute the method.
  • The present invention also relates to a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • The present invention also relates to a method for producing a food product or pharmaceutical composition.
    • BACKGROUND ART
  • The intestinal lumen is a space surrounded by a single layer of mucosal epithelial cells, and the intestinal lumen (the outside of the living body) and the inside of the living body are partitioned from each other via mucosal epithelial cells. Exposed to foreign bodies (e.g., harmful microorganisms, toxins, allergens) incorporated from the ambient environment into the intestinal lumen, the intestinal tract is an important organ in terms not only of absorption of nutrients but also of defense. The intestinal barrier function is a function to prevent foreign bodies (e.g., harmful microorganisms, toxins, allergens) incorporated from the ambient environment into the intestinal lumen from intruding into the living body.
  • The intestinal barrier function involves a physical barrier and a chemical barrier. The physical barrier is a barrier that serves literally as a physical obstacle to prevent the intrusion of foreign bodies. The physical barrier includes a mucus layer covering intestinal epithelial cells, glycocalyx, which is an assembly of sugar chains present on the surfaces of intestinal epithelial cells, and a tight junction structure, which is a cell-cell adhesion apparatus for intestinal epithelial cells. The chemical barrier is a molecular group that inhibits foreign body intrusion by exerting antibacterial activity, neutralization activity, and the like through causing chemical change to foreign bodies. The molecular group includes antibacterial peptides (e.g., defensin family molecules, Reg3 family molecules, lactoferrin, lysozyme) produced from intestinal epithelial cells such as Paneth cells. M cells, which incorporate an antigen and present it to dendritic cells, and immunoglobulin A (IgA), which is secreted into the intestinal tract to prevent foreign body intrusion by exerting neutralization activity, are each a kind of chemical barrier.
  • Reduction in intestinal barrier function causes various diseases or symptoms (e.g., enteritis, allergic diseases, psychiatric diseases, non-alcoholic fatty liver diseases, type II diabetes mellitus, metabolic syndrome, adiposity).
  • Lactulose/mannitol test (sugar tolerance test) is known as a testing method for intestinal barrier function. Intestinal permeability is tested in the lactulose/mannitol test, and increase in intestinal permeability is indicative of deterioration of intestinal barrier function (in particular, physical barrier functions including the tight junction structure). The lactulose/mannitol test has drawbacks including forcing test subjects to have long confinement time and fasting time and laying burdens on the digestive tracts of test subjects by sugar alcohol to be ingested.
  • Blood zonulin levels (Non Patent Literature 1), which are known to relate to intestinal barrier function, are used for a testing method for evaluating intestinal barrier function in some cases, but blood zonulin levels vary from day to day, and the use has problems including low reproducibility (Non Patent Literature 2).
  • No biomarker that enables simple and highly accurate evaluation of intestinal barrier function has been known.
    • CITATION LIST NON PATENT LITERATURE
    • SUMMARY OF INVENTION TECHNICAL PROBLEM
  • An object of the present invention is to provide a method for evaluating intestinal barrier function in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the diagnosis, use of a biomarker for the diagnosis, a kit for the diagnosis, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • Another object of the present invention is to provide a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the acquisition, use of a biomarker for the acquisition, a kit for the acquisition, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the screening, use of a biomarker for the screening, a kit for the screening, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • Another object of the present invention is to provide a method for producing a food product or a pharmaceutical composition.
    • SOLUTION TO PROBLEM
  • In the present invention, the substance group consisting of the substances listed in Table 1 is referred to as "the substance group of the present invention". [Table 1-1]
    Table 1 (Substance group of present invention)
    No. Substance name
    1 Melamine
    2 Azelaic acid
    3 Urocanic acid
    4 Acetanilide
    5 Paraxanthine
    6 4-Acetamidobutanoic acid
    7 Glycerol
    8 N-Acetylputrescine
    9 N8-Acetylspermidine
    10 Threonine
    11 Aminophylline
    12 2-Oxooctanoic acid
    13 Cyclohexanecarboxylic acid
    14 Muscimol
    15 3-Methoxytyrosine
    16 Theobromine
    17 Methionine sulfoxide
    18 Glyoxylic acid
    19 Serotonin
    20 Histidylglutamic acid (His-Glu)
    21 Guanidinosuccinic acid
    22 Uracil
    23 Ergothioneine
    24 Betonicine
    25 Adenine
    26 N-Methylnicotinamide
    27 4-Hexyloxyaniline
    28 Caffeine
    29 Theophylline
    30 DL-β-Leucine
    31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide
    32 Guanidineacetic acid
    33 Acetaminophen glucuronide
    34 Propyphenazone
    35 L-Threonic acid
    36 2-Ethylamino-1-phenylpropanol
    37 2-Hydroxyhippuric acid
    [Table 1-2]
    No. Substance name
    38 4-Dodecylbenzenesulfonic acid
    39 Uridine
    40 Ofloxacin
    41 (2R)-2,3-Dihydroxypropanoic acid
    42 Lactamide
  • The present invention encompasses the following inventions.
    1. [1] A method for evaluating intestinal barrier function in a subject, the method including the steps of:
      • (1a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
      • (1b) comparing the measurement value acquired in step 1a with a reference value to acquire a comparison result; and
      • (1c) evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step 1b,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    2. [2] A method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, the method including the steps of:
      • (2a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
      • (2b) comparing the measurement value acquired in step 2a with a reference value to acquire a comparison result; and
      • (2c) diagnosing the disease or symptom on the basis of the comparison result acquired in step 2b,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    3. [3] A method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject, the method including the steps of:
      • (3a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
      • (3b) comparing the measurement value acquired in step 3a with a reference value to acquire a comparison result;
      • (3c) diagnosing the disease or symptom on the basis of the comparison result acquired in step 3b; and
      • (3d) administering a substance or composition for treating the disease or symptom to the subject if the subject is diagnosed to be affected by the disease or symptom,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    4. [4] A method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function or probability of the subject being affected by the disease or symptom, the method including the steps of:
      • (4a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
      • (4b) comparing the measurement value acquired in step 4a with a reference value to acquire a comparison result; and
      • (4c) evaluating the risk or the probability on the basis of the comparison result acquired in step 4b,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    5. [5] A method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, the method including the steps of:
      • (5a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker; and
      • (5b) comparing the measurement value acquired in step 5a with a reference value to acquire a comparison result as the data,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    6. [6] A method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the method including the steps of:
      • (6a) administering a candidate substance or candidate composition to a subject having reduced intestinal barrier function;
      • (6b) measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker;
      • (6c) comparing the measurement value acquired in step 6b with a reference value to acquire a comparison result; and
      • (6d) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 6c,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    7. [7] A method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the method including the steps of:
      • (7a) administering the candidate substance or candidate composition to a subject having reduced intestinal barrier function;
      • (7b) measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker;
      • (7c) comparing the measurement value acquired in step 7b with a reference value to acquire a comparison result; and
      • (7d) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 7c,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    8. [8] The method according to any one of [1] to [7], wherein the intestinal barrier function is barrier function of a small intestine.
    9. [9] The method according to any one of [1] to [8], wherein the biomarker includes two, three, or four or more substances selected from the substance group of the present invention.
    10. [10] The method according to any one of [1] to [9], wherein the biomarker includes one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
    11. [11] The method according to [10], wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
    12. [12] The method according to any one of [1] to [9], wherein the biomarker includes one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
    13. [13] The method according to [12], wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
    14. [14] A biomarker in a specimen derived from a subject for use in evaluating intestinal barrier function in the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    15. [15] A biomarker in a specimen derived from a subject for use in diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    16. [16] A biomarker in a specimen derived from a subject for use in evaluating the subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    17. [17] A biomarker in a specimen derived from a subject for use in acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    18. [18] A biomarker in a specimen for use in screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    19. [19] A biomarker in a specimen for use in evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    20. [20] The biomarker according to any one of [14] to [19], wherein the intestinal barrier function is barrier function of a small intestine.
    21. [21] The biomarker according to any one of [14] to [20], wherein the biomarker includes two, three, or four or more substances selected from the substance group of the present invention.
    22. [22] The biomarker according to any one of [14] to [21], wherein the biomarker includes one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
    23. [23] The biomarker according to [22], wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
    24. [24] The biomarker according to any one of [14] to [21], wherein the biomarker includes one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
    25. [25] The biomarker according to [24], wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
    26. [26] Use of a biomarker in a specimen derived from a subject for evaluating intestinal barrier function in the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    27. [27] Use of a biomarker in a specimen derived from a subject for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    28. [28] Use of a biomarker in a specimen derived from a subject for evaluating the subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    29. [29] Use of a biomarker in a specimen derived from a subject for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    30. [30] Use of a biomarker in a specimen for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    31. [31] Use of a biomarker in a specimen for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    32. [32] The use according to any one of [26] to [31], wherein the intestinal barrier function is barrier function of a small intestine.
    33. [33] The use according to any one of [26] to [32], wherein the biomarker includes two, three, or four or more substances selected from the substance group of the present invention.
    34. [34] The use according to any one of [26] to [33], wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
    35. [35] The use according to [34], wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
    36. [36] The use according to any one of [26] to [33], wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
    37. [37] The use according to [36], wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
    38. [38] A kit for evaluating intestinal barrier function in a subject,
      • wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    39. [39] A kit for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject,
      • wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    40. [40] A kit for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom,
      • wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    41. [41] A kit for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject,
      • wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    42. [42] A kit for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function,
      • wherein the kit includes one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    43. [43] A kit for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function,
      • wherein the kit includes one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    44. [44] The kit according to any one of [38] to [43], wherein the intestinal barrier function is barrier function of a small intestine.
    45. [45] The kit according to any one of [38] to [44], wherein the biomarker includes two, three, or four or more substances selected from the substance group of the present invention.
    46. [46] The kit according to any one of [38] to [45], wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
    47. [47] The kit according to [46], wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
    48. [48] The kit according to any one of [38] to [45], wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
    49. [49] The kit according to [48], wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
    50. [50] A method for producing a food product or pharmaceutical composition, the method including the step of blending a substance or composition obtained through screening by the method according to [6] with one or two or more components to constitute the food product or pharmaceutical composition.
    51. [51] A computer program for allowing a computer to execute a method for evaluating intestinal barrier function in a subject, the method including the steps of:
      • (S101) acquiring a measurement value for a biomarker in a specimen derived from the subject;
      • (S102) comparing the measurement value acquired in step S101 with a reference value to acquire a comparison result; and
      • (S103) evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step S102,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    52. [52] A computer program for allowing a computer to execute a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, the method including the steps of:
      • (S201) acquiring a measurement value for a biomarker in a specimen derived from the subject;
      • (S202) comparing the measurement value acquired in step S201 with a reference value to acquire a comparison result; and
      • (S203) diagnosing the disease or symptom on the basis of the comparison result acquired in step S202,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    53. [53] A computer program for allowing a computer to execute a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, the method including the steps of:
      • (S301) acquiring a measurement value for a biomarker in a specimen derived from the subject;
      • (S302) comparing the measurement value acquired in step S301 with a reference value to acquire a comparison result; and
      • (S303) evaluating the risk or the probability on the basis of the comparison result acquired in step S302,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    54. [54] A computer program for allowing a computer to execute a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, the method including the steps of:
      • (S401) acquiring a measurement value for a biomarker in a specimen derived from the subject; and
      • (S402) comparing the measurement value acquired in step S401 with a reference value to acquire a comparison result as the data,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    55. [55] A computer program for allowing a computer to execute a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the method including the steps of:
      • (S501) acquiring a measurement value for a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject;
      • (S502) comparing the measurement value acquired in step S501 with a reference value to acquire a comparison result; and
      • (S503) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step S502,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    56. [56] A computer program for allowing a computer to execute a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the method including the steps of:
      • (S601) acquiring a measurement value for a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject;
      • (S602) comparing the measurement value acquired in step S601 with a reference value to acquire a comparison result; and
      • (S603) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step S602,
      • wherein the specimen is blood, serum, or plasma, and
      • wherein the biomarker includes one or more substances selected from the substance group of the present invention.
    57. [57] The computer program according to any one of [51] to [56], wherein the intestinal barrier function is barrier function of a small intestine.
    58. [58] The computer program according to any one of [51] to [57], wherein the biomarker includes two, three, or four or more substances selected from the substance group of the present invention.
    59. [59] The computer program according to any one of [51] to [58], wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
    60. [60] The computer program according to [59], wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
    61. [61] The computer program according to any one of [51] to [58], wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
    62. [62] The computer program according to [61], wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
    63. [63] A computer-readable recording medium storing the computer program according to any one of [51] to [62].
    ADVANTAGEOUS EFFECTS OF INVENTION
  • The present invention provides a method for evaluating intestinal barrier function in a subject, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • The present invention also provides a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the diagnosis, use of a biomarker for the diagnosis, a kit for the diagnosis, and a computer program for allowing a computer to execute the method.
  • The present invention also provides a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject.
  • The present invention also provides a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • The present invention also provides a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, a biomarker for the acquisition, use of a biomarker for the acquisition, a kit for the acquisition, and a computer program for allowing a computer to execute the method.
  • The present invention also provides a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the screening, use of a biomarker for the screening, a kit for the screening, and a computer program for allowing a computer to execute the method.
  • The present invention also provides a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, a biomarker for the evaluation, use of a biomarker for the evaluation, a kit for the evaluation, and a computer program for allowing a computer to execute the method.
  • The present invention also provides a method for producing a food product or pharmaceutical composition.
    • BRIEF DESCRIPTION OF DRAWINGS
    • Figure 1 is a schematic diagram of an apparatus according to an embodiment of the present invention.
    • Figure 2 is a block diagram illustrating the hardware configuration of an apparatus according to an embodiment of the present invention.
    • Figure 3 is a flowchart illustrating a procedure with an apparatus according to an embodiment of the present invention.
    • Figure 4 is a flowchart illustrating a procedure with an apparatus according to an embodiment of the present invention.
    • Figure 5 is a flowchart illustrating a procedure with an apparatus according to an embodiment of the present invention.
    DETAILED DESCRIPTION OF INVENTION
  • Hereinafter, the present invention will be described. Two or more of embodiments described herein can be combined, and such combinations are also included in the present invention. Herein, "E-X" indicates ×10-X, and "E+Y" indicates ×10Y.
    • <<Aspect 1>>
  • Aspect 1 of the present invention relates to a method for evaluating intestinal barrier function in a subject.
  • In the method according to Aspect 1, a biomarker in a specimen derived from the subject is used as an indicator for evaluating intestinal barrier function in the subject.
  • The subject may be any animal that has blood without limitation. Such animals include vertebrates including mammals, reptiles, birds, amphibians, and fish, mammals and birds are preferred, and mammals are more preferred. The mammals include primates (e.g., humans, gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), domestic animals (e.g., cattle, pigs, sheep, goats, horses), and pet animals (e.g., dogs, cats), and humans are preferred. The birds include poultry (e.g., chickens, wild ducks, ducks).
  • Blood derived from the subject can be obtained from the subject according to a conventional method, and the obtained blood can be used as a specimen. Serum and plasma derived from the subject can be obtained from blood obtained from the subject according to a conventional method, and the obtained serum and plasma can each be used as a specimen.
  • The blood may be blood immediately after being obtained from the subject, or blood obtained by thawing blood that have been cryopreserved immediately after being obtained from the subject. The serum and plasma may, respectively, be serum and plasma immediately after being obtained from blood, or serum and plasma obtained by thawing serum and plasma that have been cryopreserved immediately after being obtained from blood. The temperature for the cryopreservation is preferably -100°C or more and -10°C or less, more preferably -80°C or more and -15°C or less, and even more preferably -80°C or more and -60°C or less. The cryopreservation can be performed for blood, serum, or plasma with the blood, serum, or plasma contained in a container. The period of the cryopreservation is preferably 180 days or less, more preferably 60 days or less, and even more preferably 30 days or less from blood, serum, or plasma sampling. In other words, blood, serum, or plasma within 180 days from blood, serum, or plasma sampling is preferably subjected to measurement of a biomarker, blood, serum, or plasma within 60 days from blood, serum, or plasma sampling is more preferably subjected to measurement of a biomarker, and blood, serum, or plasma within 30 days from blood, serum, or plasma sampling is even more preferably subjected to measurement of a biomarker.
  • The biomarker is a substance or substance group that is present in living bodies and such that the increase or decrease of the in vivo concentration reflects the presence of a specific condition, symptom, disease, or the like, the degree of a symptom, the risk of development, the risk of being affected, a preventing effect, a therapeutic effect, an ameliorating effect, and so on. A substance group is a combination of one or more, preferably two or more substances. The biomarker is a substance or substance group to be measured in the present invention, and used as an indicator for evaluating intestinal barrier function in a subject.
  • The specimen derived from the subject is blood, serum, or plasma. Use of blood, serum, or plasma as a specimen enables simple evaluation of intestinal barrier function in the subject. The term "blood" refers to whole blood.
  • The specimen derived from the subject is preferably plasma.
  • In the case where the specimen derived from the subject is blood, serum, or plasma, one or more substances selected from the substance group of the present invention are used as the biomarker for evaluating intestinal barrier function in the subject. That is, in the case where the specimen derived from the subject is blood, serum, or plasma, the biomarker in the specimen derived from the subject includes one or more substances selected from the substance group of the present invention.
  • The biomarker in the specimen derived from the subject may include one substance selected from the substance group of the present invention, or two or more substances selected from the substance group of the present invention. For achieving enhanced accuracy of evaluating intestinal barrier function in the subject, the biomarker in the specimen derived from the subject preferably includes two, three, or four or more substances selected from the substance group of the present invention.
  • In the case where the specimen derived from the subject is blood, serum, or plasma, intestinal barrier function in the subject can be evaluated with high accuracy by using one or more substances selected from the substance group of the present invention as the biomarker. The substances of No. 1 to No. 15 and No. 27 to No. 40 in the substance group of the present invention give higher accuracy of evaluating intestinal barrier function than other substances in the substance group of the present invention, and the substances of No. 1 to No. 4 and No. 27 to No. 30 in the substance group of the present invention give particularly higher accuracy of evaluating intestinal barrier function than other substances in the substance group of the present invention.
  • In an embodiment, the specimen derived from the subject is blood, serum, or plasma, and the biomarker in the specimen derived from the subject includes one or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide). Thereby, enhanced accuracy of evaluating intestinal barrier function in the subject is achieved. In addition to one or more substances selected from the four substances, the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 5 to No. 26 in the substance group of the present invention).
  • In another embodiment, the specimen derived from the subject is blood, serum, or plasma, and the biomarker in the specimen derived from the subject includes one or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine). Thereby, enhanced accuracy of evaluating intestinal barrier function in the subject is achieved. In addition to one or more substances selected from the four substances, the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 31 to No. 42 in the substance group of the present invention).
  • In still another embodiment, the specimen derived from the subject is blood, serum, or plasma, and the biomarker in the specimen derived from the subject includes the substance of No. 2 in the substance group of the present invention (azelaic acid). Thereby, enhanced accuracy of evaluating intestinal barrier function in the subject is achieved. In addition to the one substance, the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1 and No. 3 to No. 26 in the substance group of the present invention).
  • In still another embodiment, the specimen derived from the subject is blood, serum, or plasma, and the biomarker in the specimen derived from the subject includes the substance of No. 3 in the substance group of the present invention (urocanic acid). Thereby, enhanced accuracy of evaluating intestinal barrier function in the subject is achieved. In addition to the one substance, the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1, No. 2, and No. 4 to No. 26 in the substance group of the present invention).
  • In still another embodiment, the specimen derived from the subject is blood, serum, or plasma, and the biomarker in the specimen derived from the subject includes the substance of No. 4 in the substance group of the present invention (acetanilide). Thereby, enhanced accuracy of evaluating intestinal barrier function in the subject is achieved. In addition to the one substance, the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1 to No. 3 and No. 5 to No. 26 in the substance group of the present invention).
  • In still another embodiment, the specimen derived from the subject is blood, serum, or plasma, and the biomarker in the specimen derived from the subject includes the substance of No. 5 in the substance group of the present invention (paraxanthine). Thereby, enhanced accuracy of evaluating intestinal barrier function in the subject is achieved. In addition to the one substance, the biomarker in the specimen derived from the subject may include one or more additional substances selected from the substance group of the present invention (e.g., one or more substances selected from the substances of No. 1 to No. 4 and No. 6 to No. 26 in the substance group of the present invention).
  • The method according to Aspect 1 includes the steps of:
    • (1a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker; and
    • (1b) comparing the measurement value acquired in step 1a with a reference value to acquire a comparison result; and
    • (1c) evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step 1b.
    <Step 1a>
  • Step 1a is a step of measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker.
  • The specimen derived from the subject is blood, serum, or plasma.
  • In the case where the specimen derived from the subject is blood, serum, or plasma, one or more substances selected from the substance group of the present invention is used as a biomarker for evaluating intestinal barrier function in the subject. That is, in the case where the specimen derived from the subject is blood, serum, or plasma, the biomarker to be measured in step 1a includes one or more substances selected from the substance group of the present invention.
  • In the case where the biomarker to be measured in step 1a includes one substance selected from the substance group of the present invention, the one substance is measured in step 1a. In the case where the biomarker to be measured in step 1a includes two or more substances selected from the substance group of the present invention, the two or more substances are measured in step 1a. For achieving enhanced accuracy of evaluating intestinal barrier function in the subject, it is preferable that the biomarker to be measured in step 1a includes two, three, or four or more substances selected from the substance group of the present invention (i.e., two, three, or four or more substances selected from the substance group of the present invention are measured in step 1a).
  • The phrase "measuring one substance" means measuring the concentration of the one substance, and the phrase "measuring two or more substances" means measuring the concentrations of the two or more substances, or determining a combined variable as a statistical analysis value from the concentrations of the two or more substances. Herein, a substance to be subjected to measurement (biomarker) may be referred to as a "target substance".
  • Each concentration may be a relative concentration or an absolute concentration, but is preferably an absolute concentration.
  • The relative concentration of a target substance includes a relative value that correlates with the absolute concentration of a target substance. The relative concentration of a target substance is, for example, a ratio of a measurement value for the target substance to a measurement value for an internal standard (e.g., the relative area value CE or relative area value LC described later). The relative concentration of a target substance may be a ratio given by one measurement sample, or the mean of ratios given by two or more measurement samples. Here, the two or more measurement samples are prepared from the same specimen.
  • In the present invention, "DL" (see No. 30) means a mixture of a D-form and an L-form.
  • Examples of the method for measuring a target substance include liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), gas chromatography-mass spectrometry (GC-MS), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), and CE-MS or LC-MS is preferred among these. CE-MS is suitable for measurement of water-soluble substances, and LC-MS is suitable for measurement of liposoluble substances. Particularly preferred among different types of CE-MS is capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS). Particularly preferred among different types of LC-MS is liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • Measurement samples to be used in the method for measuring a target substance can be prepared from the specimen according to a conventional method. Each measurement sample to be used in the method for measuring a target substance may contain an internal standard. The relative concentration of a target substance can be determined, for example, as a ratio of a measurement value for the target substance to a measurement value for an internal standard. The absolute concentration of a target substance can be determined, for example, from the relative concentration of the target substance with use of a pre-prepared calibration curve showing the relationship between absolute concentrations of the target substance and relative concentrations of the target substance.
  • In an embodiment, the method for measuring a target substance is CE-MS (preferably CE-FTMS). While measurement samples to be used for CE-MS can be prepared from the specimen according to a conventional method, it is preferred to prepare them from the specimen according to a method described in the Examples. While measurement by CE-MS can be performed under common conditions, it is preferred to perform the measurement under conditions described in the Examples. Each measurement sample to be used for CE-MS may contain an internal standard. Examples of the internal standard include a compound obtained by labeling a target substance with a stable isotope such as DL-phenyl-D5-alanine, a fatty acid methyl ester (e.g., methyl tetradecanoate), ribitol, and an internal standard (H3304-1002; HMT) from Human Metabolome Technologies, Inc. (HMT).
  • In the embodiment in which the method for measuring a target substance is CE-MS (preferably CE-FTMS), the relative concentration of a target substance can be determined, for example, as a ratio of the peak area acquired from the target substance in CE-MS to the peak area acquired from an internal standard in CE-MS (referred to as the "relative area value CE"). The relative concentration of a target substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples. The peak area for an internal standard can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the internal standard). The peak area for a target substance can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the target substance). The absolute concentration of a target substance can be determined, for example, from the relative concentration of the target substance with use of a pre-prepared calibration curve showing the relationship between absolute concentrations of the target substance and relative concentrations of the target substance.
  • In another embodiment, the method for measuring a target substance is LC-MS (preferably LC-MS/MS). While measurement samples to be used for LC-MS can be prepared from the specimen according to a conventional method, it is preferred to prepare them from the specimen according to a method described in the Examples. While measurement by LC-MS can be performed under common conditions, it is preferred to perform the measurement under conditions described in the Examples. Each measurement sample to be used for LC-MS may contain an internal standard. Examples of the internal standard include a compound obtained by labeling a target substance with a stable isotope such as DL-phenyl-D5-alanine, a fatty acid methyl ester (e.g., methyl tetradecanoate), ribitol, and an internal standard (H3304-1002; HMT) from Human Metabolome Technologies, Inc. (HMT).
  • In the embodiment in which the method for measuring a target substance is LC-MS (preferably LC-MS/MS), the relative concentration of a target substance can be determined, for example, as a ratio of the peak area acquired from the target substance in LC-MS to the peak area acquired from an internal standard in LC-MS (hereinafter, referred to as the "relative area value LC"). The relative concentration of a target substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples. The peak area for an internal standard can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the internal standard). The peak area for a target substance can be determined as a peak area for a compound having a specific m/z value (the compound corresponds to the target substance). The absolute concentration of a target substance can be determined, for example, from the relative concentration of the target substance with use of a pre-prepared calibration curve showing the relationship between absolute concentrations of the target substance and relative concentrations of the target substance.
  • Measurement of the substances of No. 1 to No. 26 in the substance group of the present invention is preferably performed by CE-MS (in particular, CE-FTMS), and measurement of the substances of No. 27 to No. 42 in the substance group of the present invention is preferably performed by LC-MS (in particular, LC-MS/MS). The m/z value of each substance is as shown in Table 9.
  • In the case where two or more substances (the first substance, the second substance, ..., the n-th substance (wherein n is an integer of 2 or more)) in the specimen are measured and a combined variable as a statistical analysis value is determined from the concentrations of the two or more substances in the specimen, the combined variable is preferably determined from the following expression (1): Combined variable = (Coefficient 1 × (Concentration of first substance)) + (Coefficient 2 × (Concentration of second substance)) + ... + (Coefficient n × (Concentration of n-th substance)) + Constant term ... (1)
  • In the expression (1), each "concentration" may be a relative concentration or an absolute concentration, but is preferably an absolute concentration. In the case where the method for measuring substances is CE-MS (preferably CE-FTMS), the relative concentration of each substance is, for example, a relative area value CE. The relative concentration of each substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples. In the case where the method for measuring substances is LC-MS (preferably LC-MS/MS), the relative concentration of each substance is, for example, a relative area value LC. The relative concentration of each substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • n may be any integer of 2 or more without limitation, and is, for example, an integer of 3 or more and 42 or less (e.g., 3, 4, 5, 10, 15, 20, 30, 42).
  • The coefficients and constant term depend on the method for measuring substances, the type of concentration of substances (absolute concentration or relative concentration), and the number and types of substances to be combined. The coefficients and constant term can be determined through multinomial logistic regression analysis. The coefficients are partial regression coefficients determined through multinomial logistic regression analysis. Methods for determining the coefficients and constant term through multinomial logistic regression analysis are well known to those skilled in the art. For such methods, reference can be made to documents including Metab Brain Dis, DOI 10.1007/s11011-017-0029-x, Published online: 11 May 2017, PNAS, October 14, 2003, vol. 100, no. 21, pp. 12343-12348 (www.pnas.org/cgi/doi/10.1073/pnas.2033602100, and Ann Transl Med, 2019, 7(16):388, p. 1-10 (http://dx.doi.org/10.21037/atm.2019.07.102). The coefficients to be used in determining the combined variable can be derived from measurement data in such a manner that the combined variable is close to 1 for a group with reduced intestinal barrier function, and close to 0 for a group with non-reduced intestinal barrier function (a group with normal intestinal barrier function).
  • In the case where the specimen obtained from the subject is blood, serum, or plasma, preferably plasma, the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by CE-FTMS under conditions described in the Examples, the combined variable can be determined from the concentrations of the two or more substances on the basis of the expression (1), for example, with use of coefficients and a constant term shown in Table 2. In Table 2, "CP_A" refers to melamine, "CP_B" refers to azelaic acid, "CP_C" refers to urocanic acid, and "CP_D" refers to acetanilide. [Table 2]
    Table 2 (No. 1 to No. 4 in substance group of present invention)
    Number of substances combined First substance Second substance Third substance Fourth substance Coefficient 1 Coefficient 2 Coefficient 3 Coefficient 4 Constant term
    2 CP_A CP_B - - 13467.8 -13094.0 - - -2.4228
    2 CP_A CP_C - - 14196.3 4285.6 - - -6.0130
    2 CP_A CP_D - - 11533.8 4052.7 - - -4.1778
    2 CP_B CP_C - - -18678.8 5603.3 - - -0.7664
    2 CP_B CP_D - - -11625.9 8078.4 - - -0.7713
    2 CP_C CP_D - - 3638.7 7872.2 - - -3.6483
    3 CP_A CP_B CP_C - 12507.4 -16459.5 5071.3 - -4.3082
    3 CP_A CP_B CP_D - 11896.5 -12488.3 2275.7 - -2.6231
    3 CP_A CP_C CP_D - 14738.2 4361.8 -596.1 - -6.0530
    3 CP_B CP_C CP_D - -16227.5 4956.7 3871.5 - -1.6482
    4 CP_A CP_B CP_C CP_D 16170.9 -18283.3 5697.2 -4753.2 -4.2773
  • In the case where the specimen obtained from the subject is blood, serum, or plasma, preferably plasma, the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by LC-MS/MS under conditions described in the Examples, the combined variable can be determined from the concentrations of the two or more substances on the basis of the expression (1), for example, with use of coefficients and a constant term shown in Table 3. In Table 3, "LP_A" refers to 4-hexyloxyaniline, "LP_B" refers to caffeine, "LP_C" refers to theophylline, and "LP_D" refers to DL-β-leucine. [Table 3]
    Table 3 (No. 39 to No. 42 in substance group of present invention)
    Number of substances combined First substance Second substance Third substance Fourth substance Coefficient 1 Coefficient 2 Coefficient 3 Coefficient 4 Constant term
    2 LP_A LP_B - - -1.513E-07 1.112E-07 - - 1.620613
    2 LP_A LP_C - - -1.425E-07 8.633E-09 - - 1.136544
    2 LP_A LP_D - - -1.451E-07 -4.962E-09 - - 1.966603
    2 LP_B LP_C - - -1.413E-07 1.483E-08 - - -0.81485
    2 LP_B LP_D - - 9.017E-08 -5.796E-09 - - -0.25939
    2 LP_C LP_D - - 8.573E-09 6.310E-09 - - -0.81684
    3 LP_A LP_B LP_C - -1.353E-07 -9.277E-08 1.315E-08 - 0.925891
    3 LP_A LP_B LP_D - -1.523E-07 1.187E-07 2.290E-09 - 1.538444
    3 LP_A LP_C LP_D - -1.449E-07 9.392E-09 1.387E-08 - 0.9122
    3 LP_B LP_C LP_D - -1.511E-07 1.615E-08 1.631E-08 - -1.10228
    4 LP_A LP_B LP_C LP_D -1.368E-07 -1.194E-07 1.473E-08 1.624E-08 0.793367
    • <Step 1b>
  • Step 1b is a step of comparing the measurement value acquired in step 1a with a reference value to acquire a comparison result.
  • In the case where one substance in the specimen derived from the subject is measured in step 1a, a measurement value to be acquired in step 1a is, for example, the relative concentration or absolute concentration of the one substance, and preferably the absolute concentration of the one substance. In the case where the method for measuring the one substance is CE-MS (preferably CE-FTMS), the relative concentration of the one substance is, for example, a relative area value CE. The relative concentration of the one substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples. In the case where the method for measuring the one substance is LC-MS (preferably LC-MS/MS), the relative concentration of the one substance is, for example, a relative area value LC. The relative concentration of the one substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • In the case where two or more substances in the specimen derived from the subject are measured in step 1a, measurement values to be acquired in step 1a are, for example, the relative concentrations or absolute concentrations of the two or more substances, and preferably the absolute concentrations of the two or more substances. In the case where the method for measuring the substances is CE-MS (preferably CE-FTMS), the relative concentration of each substance is, for example, a relative area value CE. The relative concentration of each substance may be a relative area value CE acquired from one measurement sample, or the mean of relative area values CE acquired from two or more measurement samples. In the case where the method for measuring the substances is LC-MS (preferably LC-MS/MS), the relative concentration of each substance is, for example, a relative area value LC. The relative concentration of each substance may be a relative area value LC acquired from one measurement sample, or the mean of relative area values LC acquired from two or more measurement samples.
  • In the case where two or more substances in the specimen derived from the subject are measured in step 1a, a measurement value to be acquired in step 1a is, for example, the combined variable determined from the concentrations of the two or more substances, and preferably the combined variable determined from the concentrations of the two or more substances on the basis of the expression (1). The concentrations to be used in determining the combined variable may be relative concentrations or absolute concentrations, but are preferably absolute concentrations.
  • In the case where one substance in the specimen derived from the subject is measured in step 1a, a measurement value for the one substance is compared with a reference value for the one substance to acquire a comparison result in step 1b.
  • In the case where the measurement value for the one substance is a relative concentration, the reference value for the one substance is also a relative concentration. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentration is determined in the same manner as for the former relative concentration. In the case where the measurement value for the one substance is a relative area value CE, the reference value for the one substance is also a relative area value CE. In the case where the measurement value for the one substance is a relative area value LC, the reference value for the one substance is also a relative area value LC.
  • In the case where the measurement value for the one substance is an absolute concentration, the reference value for the one substance is also an absolute concentration. The latter term "absolute concentration" is synonymous with the former term "absolute concentration", and the latter absolute concentration is determined in the same manner as for the former absolute concentration.
  • In the case where two or more substances in the specimen derived from the subject are measured in step 1a, measurement values for the two or more substances are compared with reference values for the two or more substances to acquire a comparison result in step 1b. For example, in the case where the substances of No. 1 and No. 2 in the substance group of the present invention are measured in step 1a, the measurement value for the substance of No. 1 is compared with the reference value for the substance of No. 1 and the measurement value for the substance of No. 2 is compared with the reference value for the substance of No. 2, thereby acquiring a comparison result in step 1b.
  • In the case where the measurement values for the two or more substances are relative concentrations, the reference values for the two or more substances are also relative concentrations. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentrations are determined in the same manner as for the former relative concentrations. In the case where the measurement values for the substances are relative area values CE, the reference values for the substances are also relative area values CE. In the case where the measurement values for the substances are relative area values LC, the reference values for the substances are also relative area values LC.
  • In the case where the measurement values for the two or more substances are absolute concentrations, the reference values for the two or more substances are also absolute concentrations. The latter term "absolute concentration" is synonymous with the former term "absolute concentration", and the latter absolute concentrations are determined in the same manner as for the former absolute concentrations.
  • In the case where a combined variable determined from the concentrations of the two or more substances (preferably, a combined variable determined from the concentrations of the two or more substances on the basis of the expression (1)) is used as a measurement value for the two or more substances, the reference value for the two or more substances is also a combined variable determined from the concentrations of the two or more substances (preferably, a combined variable determined from the concentrations of the two or more substances on the basis of the expression (1)). The latter term "combined variable" is synonymous with the former term "combined variable", and the latter combined variable is determined in the same manner as for the former combined variable.
    • <Step 1c>
  • Step 1c is a step of evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step 1b.
  • In Aspect 1, the term "positive" means that the intestinal barrier function in the subject has been reduced, and the term "negative" means that the intestinal barrier function in the subject has not been reduced.
  • The intestinal barrier function to be evaluated may be the intestinal barrier function of the small intestine or the intestinal barrier function of the large intestine, but is preferably the intestinal barrier function of the small intestine.
  • In particular, the intestinal barrier function to be evaluated is the physical barrier function including tight junction structures.
  • For substances selected from the substance group of the present invention, the intestinal barrier function in the subject can be evaluated on the basis of whether the comparison result acquired in step 1b satisfies the criterion shown in Table 4. Specifically, for each substance being "high" with respect to the criterion shown in Table 4, if the measurement value acquired in step 1a is higher than the reference value (i.e., the comparison result acquired in step 1b satisfies the criterion shown in Table 4), the case can be evaluated as being positive; if the measurement value acquired in step 1a is equal to or lower than the reference value (i.e., the comparison result acquired in step 1b does not satisfy the criterion shown in Table 4), the case can be evaluated as being negative. For each substance being "low" with respect to the criterion shown in Table 4, if the measurement value acquired in step 1a is lower than the reference value (i.e., the comparison result acquired in step 1b satisfies the criterion shown in Table 4), the case can be evaluated as being positive; if the measurement value acquired in step 1a is equal to or higher than the reference value (i.e., the comparison result acquired in step 1b does not satisfy the criterion shown in Table 4), the case can be evaluated as being negative. The criterion shown in Table 4 can also be applied to aspects other than Aspect 1.
  • In the case where one substance selected from the substance group of the present invention is measured in step 1a, if a comparison result for the one substance satisfies the criterion shown in Table 4, the case can be evaluated as being positive; if a comparison result for the one substance does not satisfy the criterion shown in Table 4, the case can be evaluated as being negative.
  • In the case where two or more substances selected from the substance group of the present invention are measured in step 1a, if a comparison result for at least one substance of the two or more substances satisfies the criterion shown in Table 4, the case can be evaluated as being positive; if none of comparison results for the two or more substances satisfies the criterion shown in Table 4, the case can be evaluated as being negative. The larger the number of substances with comparison results satisfying the criteria shown in Table 4, the higher the probability of being positive. Accordingly, two or more (e.g., two, three, or four or more) substances selected from the substance group of the present invention can be measured in step 1a to achieve enhanced accuracy of evaluating intestinal barrier function in the subject. [Table 4-1]
    Table 4 (Criterion)
    No. Substance name Criterion
    1 Melamine Low
    2 Azelaic acid High
    3 Urocanic acid Low
    4 Acetanilide Low
    5 Paraxanthine Low
    6 4-Acetamidobutanoic acid Low
    7 Glycerol High
    8 N-Acetylputrescine Low
    9 N8-Acetylspermidine Low
    10 Threonine Low
    11 Aminophylline Low
    12 2-Oxooctanoic acid Low
    13 Cyclohexanecarboxylic acid Low
    14 Muscimol Low
    15 3-Methoxytyrosine Low
    16 Theobromine Low
    17 Methionine sulfoxide Low
    18 Glyoxylic acid Low
    19 Serotonin High
    20 Histidylglutamic acid Low
    21 Guanidinosuccinic acid High
    22 Uracil High
    23 Ergothioneine High
    24 Betonicine Low
    25 Adenine High
    26 N-Methylnicotinamide High
    27 4-Hexyloxyaniline High
    28 Caffeine Low
    29 Theophylline Low
    30 DL-β-Leucine High
    31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide High
    32 Guanidineacetic acid Low
    33 Acetaminophen glucuronide High
    34 Propyphenazone High
    35 L-Threonic acid Low
    36 2-Ethylamino-1-phenylpropanol High
    37 2-Hydroxyhippuric acid High
    38 4-Dodecylbenzenesulfonic acid Low
    [Table 4-2]
    Table 4 (Continued)
    No. Substance name Criterion
    39 Uridine High
    40 Ofloxacin High
    41 (2R)-2,3-Dihydroxypropanoic acid Low
    42 Lactamide Low
  • The reference value for each substance selected from the substance group of the present invention can be set according to a conventional method.
  • In an embodiment, for each substance selected from the substance group of the present invention, the average value of measurement values for the substance in specimens obtained from a negative group (a population with non-reduced intestinal barrier function) consisting of individuals of the same species as the subject (e.g., humans if the subject is a human) can be used as a reference value for the substance. Hereinafter, this embodiment (hereinafter, referred to as "Embodiment 1-1") will be described.
  • In forming the negative group, the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test).
  • In the case where the specimen used in step 1a is blood, the specimens obtained from the negative group are also blood.
  • In the case where the specimen used in step 1a is serum, the specimens obtained from the negative group are also serum.
  • In the case where the specimen used in step 1a is plasma, the specimens obtained from the negative group are also plasma.
  • The term "measurement value" for each substance in the specimens obtained from the negative group is synonymous with that for the measurement value acquired in step 1a, and the measurement values are determined in the same manner as in step 1a.
  • In the case where the measurement value acquired in step 1a is the relative concentration of one substance selected from the substance group of the present invention, the reference value for the one substance to be used in step 1b is the average value of the relative concentrations of the one substance in the specimens obtained from the negative group. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentrations are determined in the same manner as for the former relative concentration. In the case where the former relative concentration is a relative area value CE, the latter relative concentrations are also relative area values CE. In the case where the former relative concentration is a relative area value LC, the latter relative concentrations are also relative area values LC.
  • In the case where the measurement value acquired in step 1a is the absolute concentration of one substance selected from the substance group of the present invention, the reference value for the one substance to be used in step 1b is the average value of the absolute concentrations of the one substance in the specimens obtained from the negative group. The latter term "absolute concentration" is synonymous with the former term absolute concentration, and the latter absolute concentrations are determined in the same manner as for the former absolute concentration.
  • In the case where the measurement value acquired in step 1a is the relative concentrations of two or more substances selected from the substance group of the present invention, the reference value for each substance to be used in step 1b is the average value of the relative concentrations of the substance in the specimens obtained from the negative group. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentrations are determined in the same manner as for the former relative concentrations. In the case where the former relative concentrations are relative area values CE, the latter relative concentrations are also relative area values CE. In the case where the former relative concentrations are relative area values LC, the latter relative concentrations are also relative area values LC.
  • In the case where the measurement value acquired in step 1a is the absolute concentrations of two or more substances selected from the substance group of the present invention, the reference value for each substance to be used in step 1b is the average value of the absolute concentrations of the substance in the specimens obtained from the negative group.
  • For substances selected from the substance group of the present invention, it is preferred to evaluate intestinal barrier function in the subject on the basis of whether the comparison result acquired in step 1b satisfies the criterion shown in Table 5.
  • Specifically, for a substance with a criterion of "high", a preferred criterion of x times or more, a more preferred criterion of y times or more, and an even more preferred criterion of z times or more in Table 5, if the measurement value acquired in step 1a is x times or more the average value, the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is y times or more the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is z times or more the average value, the case is even more preferably evaluated as being positive. For a substance with a criterion of "low", a preferred criterion of x times or less, a more preferred criterion of y times or less, and an even more preferred criterion of z times or less in Table 5, if the measurement value acquired in step 1a is x times or less the average value, the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is y times or less the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is z times or less the average value, the case is even more preferably evaluated as being positive.
  • Taking the substance of No. 1 in the substance group of the present invention as an example, if the measurement value acquired in step 1a is 0.9 times or less the average value, the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is 0.7 times or less the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is 0.6 times or less the average value, the case is even more preferably evaluated as being positive.
  • Taking the substance of No. 2 in the substance group of the present invention as an example, if the measurement value acquired in step 1a is 1.5 times or more the average value, the case is preferably evaluated as being positive; if the measurement value acquired in step 1a is 2.0 times or more the average value, the case is more preferably evaluated as being positive; if the measurement value acquired in step 1a is 2.5 times or more the average value, the case is even more preferably evaluated as being positive. [Table 5-1]
    Table 5 (Criterion)
    No. Substance name Criterion Preferred criterion More preferred criterion Even more preferred criterion
    1 Melamine Low 0.9 times or less 0.7 times or less 0.6 times or less
    2 Azelaic acid High 1.5 times or more 2.0 times or more 2.5 times or more
    3 Urocanic acid Low 0.9 times or less 0.7 times or less 0.5 times or less
    4 Acetanilide Low 0.9 times or less 0.7 times or less 0.5 times or less
    5 Paraxanthine Low 0.7 times or less 0.5 times or less 0.3 times or less
    6 4-Acetamidobutanoic acid Low 0.9 times or less 0.8 times or less 0.6 times or less
    7 Glycerol High 1.1 times or more 1.2 times or more 1.3 times or more
    8 N-Acetylputrescine Low 0.9 times or less 0.8 times or less 0.6 times or less
    9 N8-Acetylspermidine Low 0.9 times or less 0.8 times or less 0.6 times or less
    10 Threonine Low 0.95 times or less 0.9 times or less 0.7 times or less
    11 Aminophylline Low 0.8 times or less 0.6 times or less 0.4 times or less
    12 2-Oxooctanoic acid Low 0.9 times or less 0.7 times or less 0.5 times or less
    13 Cyclohexanecarboxylic acid Low 0.4 times or less 0.3 times or less 0.2 times or less
    14 Muscimol Low 0.9 times or less 0.8 times or less 0.6 times or less
    15 3-Methoxytyrosine Low 0.95 times or less 0.9 times or less 0.7 times or less
    16 Theobromine Low 0.9 times or less 0.7 times or less 0.5 times or less
    17 Methionine sulfoxide Low 0.9 times or less 0.8 times or less 0.6 times or less
    18 Glyoxylic acid Low 0.9 times or less 0.8 times or less 0.6 times or less
    19 Serotonin High 1.1 times or more 1.3 times or more 1.6 times or more
    20 Histidylglutamic acid Low 0.9 times or less 0.8 times or less 0.6 times or less
    21 Guanidinosuccinic acid High 1.2 times or more 1.4 times or more 2.0 times or more
    22 Uracil High 1.05 times or more 1.1 times or more 1.2 times or more
    23 Ergothioneine High 10 times or more 200 times or more 400 times or more
    [Table 5-2]
    Table 5 (Continued)
    No. Substance name Criterion Preferred criterion More preferred criterion Even more preferred criterion
    24 Betonicine Low 0.1 times or less 0.06 times or less 0.01 times or less
    25 Adenine High 8 times or more 10 times or more 15 times or more
    26 N-Methylnicotinamide High 1.05 times or more 1.1 times or more 1.2 times or more
    27 4-Hexyloxyaniline High 1.1 times or more 1.3 times or more 1.6 times or more
    28 Caffeine Low 0.8 times or less 0.6 times or less 0.4 times or less
    29 Theophylline Low 0.8 times or less 0.6 times or less 0.4 times or less
    30 DL-β-Leucine High 1.05 times or more 1.1 times or more 1.2 times or more
    31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide High 10 times or more 30 times or more 40 times or more
    32 Guanidineacetic acid Low 0.9 times or less 0.8 times or less 0.6 times or less
    33 Acetaminophen glucuronide High 10 times or more 25 times or more 30 times or more
    34 Propyphenazone High 10 times or more 200 times or more 600 times or more
    35 L-Threonic acid Low 0.95 times or less 0.9 times or less 0.7 times or less
    36 2-Ethylamino-1-phenylpropanol High 5 times or more 14 times or more 20 times or more
    37 2-Hydroxyhippuric acid High 1.05 times or more 1.1 times or more 1.2 times or more
    38 4-Dodecylbenzenesulfonic acid Low 0.9 times or less 0.8 times or less 0.6 times or less
    39 Uridine High 1.1 times or more 1.2 times or more 1.3 times or more
    40 Ofloxacin High 1.1 times or more 1.3 times or more 1.6 times or more
    41 (2R)-2,3-Dihydroxypropanoic acid Low 0.9 times or less 0.8 times or less 0.6 times or less
    42 Lactamide Low 0.95 times or less 0.9 times or less 0.7 times or less
  • In another embodiment, for each substance selected from the substance group of the present invention, the median of measurement values for the substance in specimens obtained from a negative group (a population with non-reduced intestinal barrier function) consisting of individuals of the same species as the subject (e.g., humans if the subject is a human) can be used as a reference value for the substance. Hereinafter, this embodiment (hereinafter, referred to as "Embodiment 1-2") will be described.
  • Unless otherwise specified, the description of Embodiment 1-1 is also applied to Embodiment 1-2. In the application, the term "average value" is replaced with the term "median".
  • In still another embodiment, for each substance selected from the substance group of the present invention, a cutoff value obtained from an ROC (Receiver Operating Characteristic) curve for the substance can be used as a reference value for the substance. Hereinafter, this embodiment (hereinafter, referred to as "Embodiment 1-3") will be described.
  • The ROC curve for each substance can be prepared according to a conventional method on the basis of the concentrations of the substance in specimens obtained from a negative group (a population with non-reduced intestinal barrier function) and a positive group (a population with reduced intestinal barrier function) each consisting of individuals of the same species as the subject (e.g., humans if the subject is a human). In the ROC curve, the ordinate represents sensitivity, and the abscissa represents (1 - specificity). In forming the negative group and positive group, the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test).
  • In the case where the specimen used in step 1a is blood, the specimens obtained from the negative group and positive group are also blood.
  • In the case where the specimen used in step 1a is serum, the specimens obtained from the negative group and positive group are also serum.
  • In the case where the specimen used in step 1a is plasma, the specimens obtained from the negative group and positive group are also plasma.
  • In the case where the measurement value acquired in step 1a is the relative concentration of one substance selected from the substance group of the present invention, the concentrations of the one substance to be used for preparation of the ROC curve for the one substance are also relative concentrations. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentrations are determined in the same manner as for the former relative concentrations. In the case where the former relative concentration is a relative area value CE, the latter relative concentrations are also relative area values CE. In the case where the former relative concentration is a relative area value LC, the latter relative concentrations are also relative area values LC. In the case where the concentrations to be used for preparation of the ROC curve for the one substance are relative concentrations, the cutoff value to be obtained from the ROC curve for the one substance is also a relative concentration.
  • In the case where the measurement value acquired in step 1a is the absolute concentration of one substance selected from the substance group of the present invention, the concentrations of the one substance to be used for preparation of the ROC curve for the one substance are also absolute concentrations. The latter absolute concentrations are determined in the same manner as for the former absolute concentration. In the case where the concentrations to be used for preparation of the ROC curve for the one substance are absolute concentrations, the cutoff value to be obtained from the ROC curve for the one substance is also an absolute concentration.
  • In the case where the measurement value acquired in step 1a is the relative concentrations of two or more substances selected from the substance group of the present invention, the concentrations of each substance to be used for preparation of the ROC curve for the substance are also relative concentrations. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentrations are determined in the same manner as for the former relative concentrations. In the case where the former relative concentrations are relative area values CE, the latter relative concentrations are also relative area values CE. In the case where the former relative concentrations are relative area values LC, the latter relative concentrations are also relative area values LC. In the case where the concentrations of each substance to be used for preparation of the ROC curve for the substance are relative concentrations, the cutoff value to be obtained from the ROC curve for the substance is also a relative concentration.
  • In the case where the measurement value acquired in step 1a is the absolute concentrations of two or more substances selected from the substance group of the present invention, the concentrations of each substance to be used for preparation of the ROC curve for the substance are also absolute concentrations. The latter absolute concentrations are determined in the same manner as for the former absolute concentrations. In the case where the concentrations of each substance to be used for preparation of the ROC curve are absolute concentrations, the cutoff value to be obtained from the ROC curve for the substance is also an absolute concentration.
  • A cutoff value is such a value that both high sensitivity and high specificity can be achieved when evaluation is carried out with reference to the value. A cutoff value can be appropriately set in consideration of the balance between sensitivity and specificity. For example, a value at which a high positive rate is exhibited in the positive group and a high negative rate is exhibited in the negative group can be set as a cutoff value. A method for setting a cutoff value is well known to those skilled in the art. Known examples therefor include a method in which a point on an ROC curve at which the distance from the upper left corner of the ROC curve is minimized is used as a cutoff value, and a method in which a point on an ROC curve at which the value of (sensitivity + specificity - 1) is maximized is used as a cutoff value. The point on an ROC curve at which the value of (sensitivity + specificity - 1) is maximized is called "Younden index".
  • Specific examples of cutoff values for substances selected from the substance group of the present invention are shown in Table 9.
  • The cutoff value for each substance selected from the substance group of the present invention is preferably a value within the range of a value shown as P1 in Table A or more and a value shown as P3 in Table A or less. The cutoff value for each substance selected from the substance group of the present invention may be a value within the range of a value shown as P1 in Table A or more and a value shown as P2 in Table A or less, or a value within the range of a value shown as P2 in Table A or more and a value shown as P3 in Table A or less. The cutoff value for each substance selected from the substance group of the present invention may be, for example, a value shown as P1 in Table A, or a value shown as P2 in Table A, or a value shown as P3 in Table A. [Table A-1]
    Table A
    No. Substance name P1 P2 P3
    1 Melamine 2.00E-04 2.86E-04 3.72E-04
    2 Azelaic acid 7.49E-05 1.07E-04 1.39E-04
    3 Urocanic acid 2.90E-04 4.14E-04 5.38E-04
    4 Acetanilide 1.89E-04 2.70E-04 3.51E-04
    5 Paraxanthine 7.22E-03 1.03E-02 1.34E-02
    6 4-Acetamidobutanoic acid 3.94E-05 5.63E-05 7.32E-05
    7 Glycerol 6.08E-02 8.69E-02 1.13E-01
    8 N-Acetylputrescine 1.24E-04 1.77E-04 2.30E-04
    9 N8-Acetylspermidine 5.11E-05 7.30E-05 9.50E-05
    10 Threonine 1.00E-01 1.43E-01 1.86E-01
    11 Aminophylline 1.18E-03 1.69E-03 2.20E-03
    12 2-Oxooctanoic acid 6.06E-05 8.66E-05 1.13E-04
    13 Cyclohexanecarboxylic acid 7.00E-08 1.00E-07 1.30E-07
    14 Muscimol 9.70E-05 1.39E-04 1.80E-04
    15 3-Methoxytyrosine 1.64E-04 2.34E-04 3.04E-04
    16 Theobromine 5.84E-03 8.35E-03 1.09E-02
    17 Methionine sulfoxide 1.80E-03 2.58E-03 3.35E-03
    18 Glyoxylic acid 1.89E-05 2.70E-05 3.51E-05
    19 Serotonin 1.66E-04 2.37E-04 3.08E-04
    20 Histidylglutamic acid 8.44E-06 1.21E-05 1.57E-05
    21 Guanidinosuccinic acid 5.16E-04 7.37E-04 9.58E-04
    22 Uracil 1.59E-03 2.27E-03 2.95E-03
    23 Ergothioneine 7.31E-06 1.04E-05 1.36E-05
    24 Betonicine 7.00E-08 1.00E-07 1.30E-07
    25 Adenine 1.11E-05 1.59E-05 2.07E-05
    [Table A-2]
    Table A (Continued)
    No. Substance name P1 P2 P3
    26 N-Methylnicotinamide 1.29E-06 1.84E-06 2.40E-06
    27 4-Hexyloxyaniline 8.39E+06 1.20E+07 1.56E+07
    28 Caffeine 5.75E+05 8.22E+05 1.07E+06
    29 Theophylline 4.58E+07 6.54E+07 8.50E+07
    30 DL-β-Leucine 3.24E+06 4.64E+06 6.03E+06
    31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide 6.10E+05 8.71E+05 1.13E+06
    32 Guanidineacetic acid 1.71E+07 2.45E+07 3.18E+07
    33 Acetaminophen glucuronide 1.29E+05 1.84E+05 2.39E+05
    34 Propyphenazone 4.09E+04 5.85E+04 7.60E+04
    35 L-Threonic acid 1.13E+08 1.62E+08 2.10E+08
    36 2-Ethylamino-1-phenylpropanol 1.10E+05 1.58E+05 2.05E+05
    37 2-Hydroxyhippuric acid 9.62E+04 1.37E+05 1.79E+05
    38 4-Dodecylbenzenesulfonic acid 6.29E+07 8.99E+07 1.17E+08
    39 Uridine 1.74E+08 2.48E+08 3.23E+08
    40 Ofloxacin 8.06E+04 1.15E+05 1.50E+05
    41 (2R)-2,3-Dihydroxypropanoic acid 6.55E+06 9.36E+06 1.22E+07
    42 Lactamide 1.44E+08 2.05E+08 2.67E+08
  • In still another embodiment, for two or more substances selected from the substance group of the present invention, a cutoff value for the two or more substances can be used as a reference value for the two or more substances, wherein the cutoff value is obtained in such a manner that the concentrations of the two or more substances are measured for specimens obtained from a negative group (a population with non-reduced intestinal barrier function) and a positive group (a population with reduced intestinal barrier function) each consisting of individuals of the same species as the subject (e.g., humans if the subject is a human), a combined variable is determined from the concentrations of the two or more substances on the basis of the expression (1), an ROC curve is prepared on the basis of the combined variable determined, and the cutoff value is determined from the ROC curve prepared. Hereinafter, this embodiment (hereinafter, referred to as "Embodiment 1-4") will be described.
  • Embodiment 1-4 is applied to the case where the measurement value acquired in step 1a is a combined variable determined from the concentrations of two or more substances selected from the substance group of the present invention on the basis of the expression (1).
  • In the case where the measurement value acquired in step 1a is a combined variable determined from the relative concentrations of two or more substances selected from the substance group of the present invention on the basis of the expression (1), the concentrations to be used for determination of a cutoff value are also relative concentrations. The latter term "relative concentration" is synonymous with the former term "relative concentration", and the latter relative concentrations are determined in the same manner as for the former relative concentrations. In the case where the former relative concentrations are relative area values CE, the latter relative concentrations are also relative area values CE. In the case where the former relative concentrations are relative area values LC, the latter relative concentrations are also relative area values LC.
  • In the case where the measurement value acquired in step 1a is a combined variable determined from the absolute concentrations of two or more substances selected from the substance group of the present invention on the basis of the expression (1), the concentrations to be used for determination of a cutoff value are also absolute concentrations. The latter term absolute concentration is synonymous with the former term "absolute concentration", and the latter absolute concentrations are determined in the same manner as for the former absolute concentrations.
  • In forming the negative group and positive group, the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test). An ROC curve can be prepared according to a conventional method on the basis of a combined variable obtained from the negative group and positive group. In the ROC curve, the ordinate represents sensitivity, and the abscissa represents (1 - specificity). In forming the negative group and positive group, the determination of negative and positive individuals can be performed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test). For cutoff values, the description thereof given for Embodiment 1-3 is applied.
  • In the case where the specimen is blood, serum, or plasma, preferably plasma, the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by CE-FTMS under conditions described in the Examples, specific examples of the coefficients and constant term are as shown in Table 2, and specific examples of the cutoff values are as shown in Table 6. In Table 6, "CP_A", "CP_B", "CP_C", and "CP_D" are synonymous with those in Table 2. [Table 6]
    Table 6 (No. 1 to No. 4 in substance group of present invention)
    No. Number of substances combined First substance Second substance Third substance Fourth substance Cutoff value
    1 2 CP_A CP_B - - 0.4474
    2 2 CP_A CP_C - - -0.0782
    3 2 CP_A CP_D - - 0.1073
    4 2 CP_B CP_C - - -0.1443
    5 2 CP_B CP_D - - 0.1026
    6 2 CP_C CP_D - - 0.3187
    7 3 CP_A CP_B CP_C - -0.4402
    8 3 CP_A CP_B CP_D - 0.4774
    9 3 CP_A CP_C CP_D - -0.0491
    10 3 CP_B CP_C CP_D - 0.0037
    11 4 CP_A CP_B CP_C CP_D 0.0812
  • Each of the cutoff values of No. 1 to No. 11 in Table 6 is preferably a value within the range of a value shown as Q1 in Table B or more and a value shown as Q3 in Table B or less. Each of the cutoff values of No. 1 to No. 11 in Table 6 may be a value within the range of a value shown as Q1 in Table B or more and a value shown as Q2 in Table B or less, or a value within the range of a value shown as Q2 in Table B or more and a value shown as Q3 in Table B or less. Each of the cutoff values of No. 1 to No. 11 in Table 6 may be a value shown as Q1 in Table B, or a value shown as Q2 in Table B, or a value shown as Q3 in Table B. [Table B]
    Table B
    No. Q1 Q2 Q3
    1 0.3132 0.4474 0.5816
    2 -0.1017 -0.0782 -0.0547
    3 0.0751 0.1073 0.1395
    4 -0.1876 -0.1443 -0.1010
    5 0.0718 0.1026 0.1334
    6 0.2231 0.3187 0.4143
    7 -0.5723 -0.4402 -0.3081
    8 0.3342 0.4774 0.6206
    9 -0.0638 -0.0491 -0.0344
    10 0.0026 0.0037 0.0048
    11 0.0568 0.0812 0.1056
  • In the case where the specimen is blood, serum, or plasma, preferably plasma, the biomarker to be measured in step 1a includes two or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine), and measurement of the relative area values (relative concentrations) of the two or more substances is performed by LC-MS/MS under conditions described in the Examples, specific examples of the coefficients and constant term are as shown in Table 3, and specific examples of the cutoff values are as shown in Table 7. In Table 7, "LP_A", "LP_B", "LP_C", and "LP_D" are synonymous with those in Table 3. [Table 7]
    Table 7 (No. 27 to No. 30 in substance group of present invention)
    No. Number of substances combined First substance Second substance Third substance Fourth substance Cutoff value
    1 2 LP_A LP_B - - 0.2267
    2 2 LP_A LP_C - - 0.2174
    3 2 LP_A LP_D - - 0.0855
    4 2 LP_B LP_C - - -0.1752
    5 2 LP_B LP_D - - -0.2033
    6 2 LP_C LP_D - - -0.2041
    7 3 LP_A LP_B LP_C - -0.0585
    8 3 LP_A LP_B LP_D - 0.0697
    9 3 LP_A LP_C LP_D - 0.1699
    10 3 LP_B LP_C LP_D - -0.0373
    11 4 LP_A LP_B LP_C LP_D 0.1397
  • Each of the cutoff values of No. 1 to No. 11 in Table 7 is preferably a value within the range of a value shown as R1 in Table C or more and a value shown as R3 in Table C or less. Each of the cutoff values of No. 1 to No. 11 in Table 7 may be a value within the range of a value shown as R1 in Table C or more and a value shown as R2 in Table C or less, or a value within the range of a value shown as R2 in Table C or more and a value shown as R3 in Table C or less. Each of the cutoff values of No. 1 to No. 11 in Table 7 may be a value shown as R1 in Table C, or a value shown as R2 in Table C, or a value shown as R3 in Table C. [Table C]
    Table C
    No. R1 R2 R3
    1 0.1587 0.2267 0.2947
    2 0.1522 0.2174 0.2826
    3 0.0599 0.0855 0.1112
    4 -0.2278 -0.1752 -0.1226
    5 -0.2643 -0.2033 -0.1423
    6 -0.2653 -0.2041 -0.1429
    7 -0.0761 -0.0585 -0.0410
    8 0.0488 0.0697 0.0906
    9 0.1189 0.1699 0.2209
    10 -0.0485 -0.0373 -0.0261
    11 0.0978 0.1397 0.1816
    • < <Aspect 2> >
  • Aspect 2 of the present invention relates to a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject. Hereinafter, "a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the disease or symptom".
  • The method according to Aspect 2 of the present invention includes the steps of:
    • (2a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
    • (2b) comparing the measurement value acquired in step 2a with a reference value to acquire a comparison result; and
    • (2c) diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject on the basis of the comparison result acquired in step 2b,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is also applied to Aspect 2. In the application, "step 1a" is replaced with "step 2a", "step 1b" is replaced with "step 2b", "step 1c" is replaced with "step 2c", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "diagnosing the disease or symptom in the subject", the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of diagnosing the disease or symptom in the subject", the phrase "evaluating as being positive" is replaced with the phrase "diagnosing as being positive", and the phrase "evaluating as being negative" is replaced with the phrase "diagnosing as being negative".
  • In Aspect 2, the term "positive" means that the subject is affected by the disease or symptom, and the term "negative" means that the subject is not affected by the disease or symptom.
  • The disease or symptom to be diagnosed may be a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • Examples of the disease or symptom caused by reduction in intestinal barrier function include enteritis, allergic diseases, psychiatric diseases, non-alcoholic fatty liver diseases, type II diabetes mellitus, metabolic syndrome, and adiposity.
  • Examples of the enteritis include irritable bowel syndrome, ulcerative colitis, and Crohn's disease. Examples of the allergic diseases include food allergies, atopic dermatitis, and asthma. Examples of the psychiatric diseases include anxiety disorder and depression. Examples of the non-alcoholic fatty liver diseases include non-alcoholic fatty liver and non-alcoholic steatohepatitis.
  • Diagnosis is a practice typically performed by a physician. A physician diagnoses whether the subject is affected by the disease or symptom caused by reduction in intestinal barrier function on the basis of the comparison result acquired in step 2b; if the subject is affected by the disease or symptom caused by reduction in intestinal barrier function, the physician may determine whether prevention or treatment of the disease or symptom is needed for the subject, or select a method for preventing or treating the disease or symptom for the subject, or perform prevention or treatment of the disease or symptom for the subject. The term "prevention" includes prevention, suppression, and retardation of the onset of the disease or symptom. The term "treatment" includes suppression of the progress or exacerbation of the disease or symptom, retardation of the progress or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • The method according to Aspect 2 of the present invention may be a method for assisting diagnosis of a disease or symptom caused by reduction in intestinal barrier function in a subject. The practice of assisting diagnosis of the disease or symptom is a practice of providing the comparison result acquired in step 2b as data useful for diagnosis of the disease or symptom, and this may be a medical practice or a non-medical practice, but is typically a non-medical practice. Diagnosis by a physician can be performed on the basis of the data provided through the practice of assisting diagnosis of the disease or symptom plus additional one or two or more pieces of information. Diagnosis by a physician can involve the experience, techniques, and the like of the physician.
    • < <Aspect 3> >
  • Aspect 3 of the present invention relates to a method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject. Hereinafter, "a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the disease or symptom".
  • The method according to Aspect 3 of the present invention includes the steps of:
    • (3a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
    • (3b) comparing the measurement value acquired in step 3a with a reference value to acquire a comparison result;
    • (3c) diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject on the basis of the comparison result acquired in step 3b; and
    • (3d) administering a substance or composition for treating the disease or symptom caused by reduction in intestinal barrier function to the subject if the subject is diagnosed to be affected by the disease or symptom caused by reduction in intestinal barrier function,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 2 is applied to Aspect 3. In the application, "step 2a" is replaced with "step 3a", "step 2b" is replaced with "step 3b", and "step 2c" is replaced with "step 3c".
  • For the substance or composition to be administered to the subject diagnosed to be affected by the disease or symptom caused by reduction in intestinal barrier function for treating the disease or symptom, appropriate one can be selected to fit with the type of the disease or symptom caused by reduction in intestinal barrier function by which the subject is affected. The term "treatment" includes suppression of the progression or exacerbation of a disease or symptom, retardation of the progression or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • Substances or compositions for treating irritable bowel syndrome include high-molecular-weight polymers and serotonin receptor antagonists. Substances or compositions for treating ulcerative colitis or Crohn's disease include 5-aminosalicylate preparations and corticosteroids. Substances or compositions for treating allergic diseases include antihistamines. Substances or compositions for treating anxiety disorder or depression include selective serotonin reuptake inhibitors and noradrenergic antidepressants. Substances or compositions for treating non-alcoholic fatty liver diseases include antioxidants, therapeutics for diabetes mellitus, and therapeutics for dyslipidemia.
  • The substance or composition for treating the disease or symptom may be administered in the form of a food composition containing the substance or composition. Examples of the food composition include yogurt, probiotics, prebiotics, and supplements. The food composition may be, for example, a food for specified health uses, a food with functional claims, or a food for medical use.
  • Examples of the route of administration of the substance or composition for treating the disease or symptom include oral and parenteral (e.g., intranasal, ophthalmic, ear-drop, transdermal, tracheobronchial, intrarectal, urinary, subcutaneous, intramuscular, intravenous) administrations. The substance or composition for treating the disease or symptom may be formulated into a dosage form suitable for the route of administration. Examples of the dosage form include a tablet and an injection. In formulating, a proper excipient for oral administration (e.g., a diluent, a disintegrant, a lubricant, a binder) or an excipient suitable for parenteral administration (e.g., a diluting agent, a solvent) can be used. The dose and frequency of administration of the substance or composition for treating the disease or symptom can be appropriately adjusted in consideration of the dosage form and the age, body weight, and so on of the subject. The frequency of administration per day may be once, or two or more times. The period of administration may be 1 day or 2 days or more, (e.g., 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more).
    • < <Aspect 4> >
  • Aspect 4 of the present invention relates to a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom in a subject. Hereinafter, "a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the risk of the development", and "probability of the subject being affected by a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the probability of being affected".
  • The method according to Aspect 4 includes the steps of:
    • (4a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker; and
    • (4b) comparing the measurement value acquired in step 4a with a reference value to acquire a comparison result; and
    • (4c) evaluating the risk of the development or the probability of being affected on the basis of the comparison result acquired in step 4b,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 4. In the application, "step 1a" is replaced with "step 4a", "step 1b" is replaced with "step 4b", "step 1c" is replaced with "step 4c", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "evaluating the risk of the development or the probability of being affected", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of evaluating the risk of the development or the probability of being affected".
  • In Aspect 4, the term "positive" means that the risk of the development or the probability of being affected is present or high, and the term "negative" means that the risk of the development or the probability of being affected is absent or low.
  • The risk of the development to be evaluated may be a subject's risk of developing a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a subject's risk of developing a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a subject's risk of developing a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • The probability of being affected to be evaluated may be probability of the subject being affected by a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or probability of the subject being affected by a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably probability of the subject being affected by a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • For the disease or symptom caused by reduction in intestinal barrier function, the description thereof given for Aspect 2 is applied.
    • < <Aspect 5> >
  • Aspect 5 of the present invention relates to a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject. Hereinafter, "a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the disease or symptom".
  • The method according to Aspect 5 includes the steps of:
    • (5a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker; and
    • (5b) comparing the measurement value acquired in step 5a with a reference value to acquire a comparison result as the data for diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 5. In the application, "step 1a" is replaced with "step 5a", "step 1b" is replaced with "step 5b", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "acquiring data for diagnosing the disease or symptom in the subject", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of diagnosing the disease or symptom in the subject".
  • In Aspect 5, the term "positive" means that the subject is affected by the disease or symptom, and the term "negative" means that the subject is not affected by the disease or symptom.
  • The disease or symptom to be evaluated may be a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • For the disease or symptom caused by reduction in intestinal barrier function, the description thereof given for Aspect 2 is applied.
  • For substances selected from the substance group of the present invention, a comparison result satisfying the criterion shown in Table 4 can be acquired as data for diagnosing as being positive, and a comparison result not satisfying the criterion shown in Table 4 can be acquired as data for diagnosing as being negative. Specifically, for a substance being "high" with respect to the criterion shown in Table 4, a comparison result that the measurement value acquired in step 5a is higher than the reference value can be acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is equal to or lower than the reference value can be acquired as data for diagnosing as being negative. For a substance being "low" with respect to the criterion shown in Table 4, a comparison result that the measurement value acquired in step 5a is lower than the reference value can be acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is equal to or higher than the reference value can be acquired as data for diagnosing as being negative.
  • In the case where one substance selected from the substance group of the present invention is measured in step 5a, if a comparison result for the one substance satisfies the criterion shown in Table 4, the comparison result can be acquired as data for diagnosing as being negative; if a comparison result for the one substance does not satisfy the criterion shown in Table 4, the comparison result can be acquired as data for diagnosing as being negative.
  • In the case where two or more substances selected from the substance group of the present invention are measured in step 5a, if a comparison result for at least one substance of the two or more substances satisfies the criterion shown in Table 4, the comparison result can be acquired as data for diagnosing as being positive; if none of comparison results for the two or more substances satisfies the criterion shown in Table 4, the comparison results can be acquired as data for diagnosing as being negative. The larger the number of substances with comparison results satisfying the criteria shown in Table 4, the higher the probability of being positive. Accordingly, two or more (e.g., two, three, or four or more) substances selected from the substance group of the present invention can be measured in step 5a to achieve enhanced accuracy of diagnosing a disease or symptom in a subject.
  • In the case where Embodiment 1-1 of Aspect 1 is applied to Aspect 5, for each substance selected from the substance group of the present invention, a comparison result based on the criterion shown in Table 5 is preferably acquired as data for diagnosis.
  • Specifically, for a substance with a criterion of "high", a preferred criterion of x times or more, a more preferred criterion of y times or more, and an even more preferred criterion of z times or more in Table 5, a comparison result that the measurement value acquired in step 5a is x times or more the average value is preferably acquired as data for diagnosing as being positive, a comparison result that the measurement value acquired in step 5a is y times or more the average value is more preferably acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is z times or more the average value is even more preferably acquired as data for diagnosing as being positive. For a substance with a criterion of "low", a preferred criterion of x times or less, a more preferred criterion of y times or less, and an even more preferred criterion of z times or less in Table 5, a comparison result that the measurement value acquired in step 5a is x times or less the average value is preferably acquired as data for diagnosing as being positive, a comparison result that the measurement value acquired in step 5a is y times or less the average value is more preferably acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is z times or less the average value is even more preferably acquired as data for diagnosing as being positive.
  • Taking the substance of No. 1 in the substance group of the present invention as an example, a comparison result that the measurement value acquired in step 5a is 0.9 times or less the average value is preferably acquired as data for diagnosing as being positive, a comparison result that the measurement value acquired in step 5a is 0.7 times or less the average value is more preferably acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is 0.6 times or less the average value is even more preferably acquired as data for diagnosing as being positive.
  • Taking the substance of No. 2 in the substance group of the present invention as an example, a comparison result that the measurement value acquired in step 5a is 1.5 times or more the average value is preferably acquired as data for diagnosing as being positive, a comparison result that the measurement value acquired in step 5a is 2.0 times or more the average value is more preferably acquired as data for diagnosing as being positive, and a comparison result that the measurement value acquired in step 5a is 2.5 times or more the average value is even more preferably acquired as data for diagnosing as being positive.
  • The data acquired in step 5b can be used for diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject, or for assisting diagnosis of the disease or symptom caused by reduction in intestinal barrier function in the subject.
  • Diagnosis is a practice typically performed by a physician. A physician diagnoses whether the subject is affected by the disease or symptom caused by reduction in intestinal barrier function on the basis of the data acquired in step 5b; if the subject is affected by the disease or symptom caused by reduction in intestinal barrier function, the physician may determine whether prevention or treatment of the disease or symptom is needed for the subject, or select a method for preventing or treating the disease or symptom for the subject, or perform prevention or treatment of the disease or symptom for the subject. The term "prevention" includes prevention, suppression, and retardation of the onset of the disease or symptom. The term "treatment" includes suppression of the progress or exacerbation of the disease or symptom, retardation of the progress or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • The practice of assisting diagnosis of the disease or symptom is a practice of providing the data acquired in step 5b as data for diagnosis of the disease or symptom, and this may be a medical practice or a non-medical practice, but is typically a non-medical practice. Diagnosis by a physician can be performed on the basis of the data provided through the practice of assisting diagnosis of the disease or symptom plus additional one or two or more data.
    • <<Aspect 6> >
  • Aspect 6 of the present invention relates to a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function. Hereinafter, the phrase "a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the preventing or ameliorating effect on the disease or symptom".
  • The method according to Aspect 6 includes the steps of:
    • (6a) administering a candidate substance or candidate composition to a subject having reduced intestinal barrier function;
    • (6b) measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker; and
    • (6c) comparing the measurement value acquired in step 6b with a reference value to acquire a comparison result; and
    • (6d) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 6c,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 6. In the application, "step 1a" is replaced with "step 6b", "step 1b" is replaced with "step 6c", "step 1c" is replaced with "step 6d", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom".
  • In Aspect 6, the term "positive" means that the candidate substance or candidate composition has the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom, and the term "negative" means that the candidate substance or candidate composition does not have the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom. The term "prevention" includes prevention, suppression, and retardation of the onset of the disease or symptom. The term "amelioration" includes suppression of the progression or exacerbation of the disease or symptom, retardation of the progression or exacerbation of the disease or symptom, and relief, mitigation, amelioration, and cure of the disease or symptom.
  • The improving effect on intestinal barrier function to be evaluated may be an improving effect on the intestinal barrier function of the small intestine, or an improving effect on the intestinal barrier function of the large intestine, but is preferably an improving effect on the intestinal barrier function of the small intestine.
  • The preventing or ameliorating effect on the disease or symptom to be evaluated may be a preventing or ameliorating effect on a disease or symptom caused by reduction in the intestinal barrier function of the small intestine, or a preventing or ameliorating effect on a disease or symptom caused by reduction in the intestinal barrier function of the large intestine, but is preferably a preventing or ameliorating effect on a disease or symptom caused by reduction in the intestinal barrier function of the small intestine.
  • For the disease or symptom caused by reduction in intestinal barrier function, the description thereof given for Aspect 2 is applied.
  • The method according to Aspect 6 allows screening to obtain a candidate substance or candidate composition evaluated to have the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom as a substance or composition having the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • Step 6a is a step of administering a candidate substance or candidate composition to a subject having reduced intestinal barrier function.
  • The reduced intestinal barrier function in the subject can be confirmed by a known testing method for intestinal barrier function (e.g., lactulose/mannitol test), or by the method according to Aspect 1.
  • The method according to Aspect 6 may include a step of evaluating intestinal barrier function in a subject to confirm reduced intestinal barrier function in the subject by the method according to Aspect 1 before step 6a. In the case where the method according to Aspect 6 includes this step, the biomarker to be measured in step 1a in Aspect 1 may be the same as or different from the biomarker to be measured in step 6b in terms of type, but they are preferably the same for enhanced accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • The method according to Aspect 6 may include a step of selecting a subject having reduced intestinal barrier function by the method according to Aspect 1 before step 6a. In the case where the method according to Aspect 6 includes this step, the biomarker to be measured in step 1a in Aspect 1 may be the same as or different from the biomarker to be measured in step 6b in terms of type, but they are preferably the same for enhanced accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • The subject to which the candidate substance or candidate composition is to be administered is not limited as long as the intestinal barrier function has been reduced, and may be a human having reduced intestinal barrier function or a model animal (except humans) having reduced intestinal barrier function, but is preferably a model animal (except humans) having reduced intestinal barrier function. Such model animals include vertebrates including mammals, reptiles, birds, amphibians, and fish, mammals and birds are preferred, and mammals are more preferred. The mammals include primates (e.g., gorillas, chimpanzees, orangutans), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), domestic animals (e.g., cattle, pigs, sheep, goats, horses), and pet animals (e.g., dogs, cats). The birds include poultry (e.g., chickens, wild ducks, ducks).
  • The candidate substance or candidate composition to be administered to the subject having reduced intestinal barrier function is not limited, and can be selected from, for example, high-molecular-weight compounds, low-molecular-weight compounds, cell cultures, cell extracts, antibodies, proteins, peptides, nucleic acids, carbohydrates, inorganic salts, metal complexes, microorganisms (bacteria (probiotics), yeasts, etc.), dietary fibers, resistant carbohydrates, resistant proteins, fermented products (including fermented foods), prebiotics, and any combination of two or more of these.
  • Examples of the route of administration of the candidate substance or candidate composition to the subject include oral and parenteral (e.g., intranasal, ophthalmic, ear-drop, transdermal, tracheobronchial, intrarectal, urinary, subcutaneous, intramuscular, intravenous) administrations. The candidate substance or candidate composition may be formulated into a dosage form suitable for the route of administration. Examples of the dosage form include a tablet and an injection. In formulating, a proper excipient for oral administration (e.g., a diluent, a disintegrant, a lubricant, a binder) or an excipient suitable for parenteral administration (e.g., a diluting agent, a solvent) can be used. The dose and frequency of administration of the candidate substance or candidate composition can be appropriately adjusted in consideration of the dosage form and the age, body weight, and so on of the subject. The frequency of administration per day may be once, or two or more times. The period of administration may be 1 day or 2 days or more (e.g., 1 week or more, 2 weeks or more, 3 weeks or more, or 4 weeks or more).
  • Step 6b is a step of measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker.
  • Step 6b can be performed in the same manner as step 1a in Aspect 1, except that a specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject is used.
  • The specimen to be used in step 6b is obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject. The specimen may be obtained from the subject at multiple time points after administration of the candidate substance or candidate composition. The time point to obtain the specimen can be appropriately adjusted in consideration of the dosage form and the age, body weight, and so on of the subject. In an embodiment, the time point to obtain the specimen is, for example, any time point between 0.5 and 24 hours after administration, or any time point between 0.5 and 12 hours after administration, or any time point between 1 and 10 hours after administration. Here, the meaning of "after administration" may be "after one administration" or "after two or more continuous administrations". The meaning of "after two or more continuous administrations" may be, for example, "after the final administration of continuous administrations for 4 weeks or more", or "after the final administration of continuous administrations for 1 day to 4 weeks", or "after the final administration of continuous administrations for 3 days to 3 weeks", or "after the final administration of continuous administrations for 1 week to 2 weeks".
  • Step 6c is a step of comparing the measurement value acquired in step 6b with a reference value to acquire a comparison result.
  • Step 6c can be performed in the same manner as step 1b in Aspect 1, except that the measurement value acquired in step 6b is used.
  • Step 6d is a step of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 6c.
  • Step 6d can be performed in the same manner as step 1c in Aspect 1, except that the candidate substance or candidate composition is evaluated for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
  • For substances selected from the substance group of the present invention, the candidate substance or candidate composition can be evaluated for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of whether the comparison result acquired in step 6c satisfies the criterion shown in Table 4. Specifically, for a substance being "high" with respect to the criterion shown in Table 4, if the measurement value acquired in step 6b is higher than the reference value (i.e., the comparison result acquired in step 6c satisfies the criterion shown in Table 4), the case can be evaluated as being negative; if the measurement value acquired in step 6b is equal to or lower than the reference value (i.e., the comparison result acquired in step 6c does not satisfy the criterion shown in Table 4), the case can be evaluated as being positive. For a substance being "low" with respect to the criterion shown in Table 4, if the measurement value acquired in step 6b is lower than the reference value (i.e., the comparison result acquired in step 6c satisfies the criterion shown in Table 4), the case can be evaluated as being negative; if the measurement value acquired in step 6b is equal to or higher than the reference value (i.e., the comparison result acquired in step 6c does not satisfy the criterion shown in Table 4), the case can be evaluated as being positive.
  • In the case where one substance selected from the substance group of the present invention is measured in step 6b, if a comparison result for the one substance satisfy the criterion shown in Table 4, the case can be evaluated as being negative; if a comparison result for the one substance does not satisfy the criterion shown in Table 4, the case can be evaluated as being positive.
  • In the case where two or more substances selected from the substance group of the present invention are measured in step 6b, if all comparison results for the two or more substances satisfy the criteria shown in Table 4, the case can be evaluated as being negative; if a comparison result for at least one substance of the two or more substances does not satisfy the criterion shown in Table 4, the case can be evaluated as being positive. The larger the number of substances with comparison results not satisfying the criteria shown in Table 4, the higher the probability of being positive. Accordingly, two or more (e.g., two, three, or four or more) substances selected from the substance group of the present invention can be measured in step 6b to achieve enhanced accuracy of evaluating a candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom.
    • < <Aspect 7> >
  • Aspect 7 of the present invention relates to a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function. Hereinafter, the phrase "a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the preventing or ameliorating effect on the disease or symptom".
  • The method according to Aspect 7 includes the steps of:
    • (7a) administering a candidate substance or candidate composition to a subject having reduced intestinal barrier function;
    • (7b) measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker; and
    • (7c) comparing the measurement value acquired in step 7b with a reference value to acquire a comparison result; and
    • (7d) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 7c,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 6 is applied to Aspect 7. In the application, "step 6a" is replaced with "step 7a", "step 6b" is replaced with "step 7b", "step 6c" is replaced with "step 7c", and "step 6d" is replaced with "step 7d".
    • <<Aspect 8>>
  • Aspect 8 of the present invention relates to a biomarker in a specimen derived from a subject for use in evaluating intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 8.
    • <<Aspect 9>>
  • Aspect 9 of the present invention relates to a biomarker in a specimen derived from a subject for use in diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 2 is applied to Aspect 9.
    • < <Aspect 10> >
  • Aspect 10 of the present invention relates to a biomarker in a specimen derived from a subject for use in evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 4 is applied to Aspect 10.
    • < <Aspect 11 > >
  • Aspect 11 of the present invention relates to a biomarker in a specimen derived from a subject for use in acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 5 is applied to Aspect 11.
    • <<Aspect 12>>
  • Aspect 12 of the present invention relates to a biomarker in a specimen for use in screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 6 is applied to Aspect 12.
    • <<Aspect 13>>
  • Aspect 13 of the present invention relates to a biomarker in a specimen for use in evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 7 is applied to Aspect 13.
    • < <Aspect 14> >
  • Aspect 14 of the present invention relates to use of a biomarker in a specimen derived from a subject for evaluating intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 14.
    • <<Aspect 15>>
  • Aspect 15 of the present invention relates to use of a biomarker in a specimen derived from a subject for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 2 is applied to Aspect 15.
    • < <Aspect 16> >
  • Aspect 16 of the present invention relates to use of a biomarker in a specimen derived from a subject for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 4 is applied to Aspect 16.
    • < <Aspect 17> >
  • Aspect 17 of the present invention relates to use of a biomarker in a specimen derived from a subject for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 5 is applied to Aspect 17.
    • < <Aspect 18 > >
  • Aspect 18 of the present invention relates to use of a biomarker in a specimen for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 6 is applied to Aspect 18.
    • < <Aspect 19> >
  • Aspect 19 of the present invention relates to use of a biomarker in a specimen for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 7 is applied to Aspect 19.
    • < <Aspect 20> >
  • Aspect 20 of the present invention relates to a kit for evaluating intestinal barrier function in a subject, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 20.
  • The kit may include one reagent, or two or more reagents.
  • A reagent of any type that fits with the type of method for measuring a target substance can be appropriately selected. Examples of the method for measuring a target substance include liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), gas chromatography-mass spectrometry (GC-MS), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), and CE-MS or LC-MS is preferred among these. CE-MS is suitable for measurement of water-soluble substances, and LC-MS is suitable for measurement of liposoluble substances. Particularly preferred among different types of CE-MS is capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS). Particularly preferred among different types of LC-MS is liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • In an embodiment, the kit includes one or more reagents for measuring the concentration of a target substance by CE-MS. The concentration may be a relative concentration or an absolute concentration, but is preferably an absolute concentration. The relative concentration is, for example, a relative area value CE. Examples of the reagent for measuring the concentration of a target substance by CE-MS include authentic samples of one or more substances selected from the substance group of the present invention.
  • In another embodiment, the kit include one or more reagents for measuring the concentration of a target substance by LC-MS. The concentration may be a relative concentration or an absolute concentration, but is preferably an absolute concentration. The relative concentration is, for example, a relative area value LC. Examples of the reagent for measuring the concentration of a target substance by LC-MS include authentic samples of one or more substances selected from the substance group of the present invention.
    • < <Aspect 21> >
  • Aspect 21 of the present invention relates to a kit for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 2 is applied to Aspect 21.
  • For the reagents, the description thereof given for Aspect 20 is applied.
    • < <Aspect 22> >
  • Aspect 22 of the present invention relates to a kit for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 4 is applied to Aspect 22.
  • For the reagents, the description thereof given for Aspect 20 is applied.
    • < <Aspect 23> >
  • Aspect 23 of the present invention relates to a kit for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, wherein the kit includes one or more reagents for measuring a biomarker in a specimen derived from the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 5 is applied to Aspect 23.
  • For the reagents, the description thereof given for Aspect 20 is applied.
    • <<Aspect 24>>
  • Aspect 24 of the present invention relates to a kit for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, wherein the kit includes one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 6 is applied to Aspect 24.
  • For the reagents, the description thereof given for Aspect 20 is applied.
    • < <Aspect 25> >
  • Aspect 25 of the present invention relates to a kit for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, wherein the kit includes one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject, wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 7 is applied to Aspect 25.
  • For the reagents, the description thereof given for Aspect 20 is applied.
    • <<Aspect 26>>
  • Aspect 26 of the present invention relates to a method for producing a food product or pharmaceutical composition, the method including the step of blending a substance or composition obtained through screening by the method according to Aspect 6 with one or two or more components to constitute the food product or pharmaceutical composition.
  • The one or two or more components to be blended with the substance or composition obtained through screening by the method according to Aspect 6 may be any ones acceptable as components for a food product or pharmaceutical composition without limitation, and appropriate ones can be selected to fit with the type of food product or pharmaceutical composition to be produced.
  • The method according to Aspect 26 may include a step of screening for a substance or composition by the method according to Aspect 6.
    • < <Aspect 27> >
  • Aspect 27 of the present invention relates to a computer program for allowing a computer to execute a method for evaluating intestinal barrier function in a subject.
  • The method that a computer is allowed to execute by the computer program according to Aspect 27 includes the steps of:
    • (S101) acquiring a measurement value for a biomarker in a specimen derived from the subject;
    • (S102) comparing the measurement value acquired in step S101 with a reference value to acquire a comparison result; and
    • (S103) evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step S102,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 1 is applied to Aspect 27.
  • The computer program according to Aspect 27 allows a computer to execute a method for evaluating intestinal barrier function in a subject when the computer program is executed by the computer. The computer program according to Aspect 27 includes an instruction to allow a computer to execute a method for evaluating intestinal barrier function in a subject.
  • Hereinafter, an embodiment of Aspect 27 will be described with reference to drawings. Figure 1 is a schematic diagram of an apparatus to be used in the present embodiment, Figure 2 is a block diagram illustrating the hardware configuration of the apparatus of the present embodiment, and Figures 3 to 5 are each a flowchart illustrating a procedure with the apparatus of the present embodiment.
  • As illustrated in Figure 1, an apparatus 10 includes a computer system 30. As illustrated in Figure 1, the apparatus 10 may include a measurement device 20 connected to the computer system 30. The computer system 30 may be a system provided separately from the measurement device 20, or a system including the measurement device 20 therein. The apparatus 10 may be an apparatus in which the measurement device 20 and the computer system 30 are integrally configured.
  • The measurement device 20 executes measurement of a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker (e.g., an absolute concentration, a relative concentration, or a combined variable) or a measurement value necessary for calculating a measurement value for the biomarker (e.g., a measurement value necessary for calculating a relative concentration or a combined variable), and sends the acquired value to the computer system 30. For the specimen derived from the subject, the measurement of the biomarker, and the measurement value for the biomarker, the description thereof given for Aspect 1 is applied.
  • For example, the measurement device 20 is an automated measurement device that executes measurement of the biomarker by CE-MS (in particular, CE-FTMS) or LC-MS (in particular, LC-MS/MS) to acquire a measurement value for the biomarker (e.g., a relative area value CE, a relative area value LC, or a combined variable) or a measurement value necessary for calculating a measurement value for the biomarker (e.g., a measurement value necessary for calculating a relative area value CE, a relative area value LC, or a combined variable).
  • The measurement device 20 is needed to be able to execute measurement of the biomarker in a measurement sample (e.g., a measurement sample to be used for CE-MS (in particular, CE-FTMS) or LC-MS (in particular, LC-MS/MS)) and the subsequent process. Processes prior to measurement of the biomarker in a measurement sample such as collection of blood, serum, or plasma derived from the subject (i.e., collection of blood, serum, or plasma to be used as a specimen from blood obtained from the subject), preparation of a measurement sample from blood, serum, or plasma derived from the subject, and setting of a measurement sample to the measurement device 20 may be executed by a human or by the measurement device 20. In an embodiment, processes prior to measurement of the biomarker in a measurement sample are executed by a human.
  • As illustrated in Figure 2, the computer system 30 includes: a computer main body 300; an input section 301; and a display section 302 that displays specimen information, evaluation results, and so on.
  • Based on the measurement value for the biomarker, a processor of the computer system 30 executes the computer program, installed in a hard disk 313, for evaluating intestinal barrier function in the subject.
  • As illustrated in Figure 2, the computer main body 300 includes: a CPU (Central Processing Unit) 310; a ROM (Read Only Memory) 311; a RAM (Random Access Memory) 312; a hard disk 313; an input/output interface 314; a reader 315; a communication interface 316; and an image output interface 317. The CPU 310, ROM 311, RAM 312, hard disk 313, input/output interface 314, reader 315, communication interface 316, and image output interface 317 are connected together via buses 318 in a manner that allows data communication. The measurement device 20 is communicatively connected to the computer system 30 via the communication interface 316.
  • The CPU 310 is capable of executing a program stored in the ROM 311 or hard disk 313 or a program loaded in the RAM 312. Through execution of a program by the CPU 310, the apparatus 10 functions as an apparatus for evaluating intestinal barrier function in the subject.
  • The ROM 311 is configured with a mask ROM, a PROM, an EPROM, an EEPROM, or the like. A computer program to be executed by the CPU 310 and data to be used for execution of the computer program are stored in the ROM 311.
  • The RAM 312 is configured with an SRAM, a DRAM, or the like. The RAM 312 is used for reading programs stored in the ROM 311 and hard disk 313. In addition, the RAM 312 is used as a working area for the CPU 310 when a program is executed.
  • In the hard disk 313, an operating system to be executed by the CPU 310, computer programs including application programs, and data to be used for execution of the computer programs have been installed.
  • The reader 315 is configured with a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a memory stick reader, a solid state drive, or the like. The reader 315 is capable of reading programs or data stored in a transportable storage medium 400.
  • The input/output interface 314 is configured with a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE 1284, and an analog interface consisting of a D/A converter, an A/D converter, or the like. To the input/output interface 314, the input section 301 including a keyboard and a mouse is connected. An operator can input various instructions to the computer main body 300 via the input section 301.
  • The communication interface 316 is, for example, an Ethernet (R) interface. The computer main body 300 is capable of executing sending of print data, for example, to a printer, a computer of the subject or a service provider, or a smartphone terminal of the subject through the communication interface 316.
  • The image output interface 317 is connected to the display section 302 configured with an LCD, a CRT, or the like. With this configuration, the display section 302 is capable of outputting video signals corresponding to image data given by the CPU 310. The display section 302 displays an image (picture) according to inputted video signals. The display section 302 may display raw data themselves, or an illustration, a drawing, or the like made by converting raw data for easy understanding by the subject.
  • Hereinafter, a procedure with the apparatus 10 will be described with reference to Figures 3 to 5.
  • As illustrated in Figure 3, in step S101, the CPU 310 acquires a measurement value for a biomarker in a specimen derived from the subject. For the specimen derived from the subject and the measurement value for the biomarker, the description thereof given for Aspect 1 is applied.
  • In an embodiment, the measurement device 20 executes measurement of a biomarker in a specimen derived from the subject, acquires a measurement value for the biomarker (e.g., an absolute concentration, a relative concentration, or a combined variable), and sends the acquired value to the computer system 30. For the specimen derived from the subject, the measurement of the biomarker, and the measurement value for the biomarker, the description thereof given for Aspect 1 is applied. The CPU 310 stores the measurement value for the biomarker, the measurement value sent from the measurement device 20, in the hard disk 313. In step S301, the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • In another embodiment, the measurement device 20 executes measurement of a biomarker in a specimen derived from the subject, acquires a measurement value necessary for calculating a measurement value for the biomarker (e.g., a measurement value necessary for calculating a relative concentration or a combined variable), and sends the acquired value to the computer system 30. For the specimen derived from the subject, the measurement of the biomarker, and the measurement value for the biomarker, the description thereof given for Aspect 1 is applied. Based on the measurement value sent from the measurement device 20, the CPU 310 calculates a measurement value for the biomarker (e.g., a relative concentration or a combined variable), and stores the measurement value for the biomarker in the hard disk 313. In step S301, the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • In still another embodiment, an operator inputs a measurement value for a biomarker (e.g., an absolute concentration, a relative concentration, or a combined variable), the measurement value acquired in advance through measurement of the biomarker in a specimen derived from the subject, via the input section 301. For the specimen derived from the subject, the measurement of the biomarker, and the measurement value for the biomarker, the description thereof given for Aspect 1 is applied. The CPU 310 stores the inputted measurement value for the biomarker in the hard disk 313. In step S301, the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • In still another embodiment, an operator inputs a measurement value necessary for calculating a measurement value for a biomarker (e.g., a measurement value necessary for calculating a relative concentration or a combined variable), the measurement value acquired in advance through measurement of the biomarker in a specimen derived from the subject, via the input section 301. For the specimen derived from the subject, the measurement of the biomarker, and the measurement value for the biomarker, the description thereof given for Aspect 1 is applied. Based on the inputted measurement value, the CPU 310 calculates a measurement value for the biomarker (e.g., a relative concentration or a combined variable), and stores the measurement value for the biomarker in the hard disk 313. In step S301, the CPU 310 acquires the measurement value for the biomarker, the measurement value stored in the hard disk 313.
  • As illustrated in Figure 3, in step S102, the CPU 310 compares the measurement value for the biomarker with a reference value stored in the hard disk 313 to acquire a comparison result. For the reference value, the comparison, and the comparison result, the description thereof given for Aspect 1 is applied. The CPU 310 stores the acquired comparison result in the hard disk 313. The CPU 310 may output the acquired comparison result to allow the display section 302 to display the comparison result, or allow a printer to print the comparison result, or transfer the comparison result to another computer or an application (such as a smartphone). In outputting the comparison result, the CPU 310 may allow the display section 302 to display the measurement value for the biomarker, the reference value, and others as reference information, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone). Together with the comparison result, the CPU 310 may allow the display section 302 to display advice on life, foods recommended to ingest (including functional foods), and others for improved intestinal barrier function, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone). Thereby, an index for evaluating intestinal barrier function in the subject can be provided, for example, to a physician, a nurse, a caregiver, a tester, a testing business operator, a service provider, or the subject himself/herself.
  • As illustrated in Figure 3, in step S103, the CPU 310 evaluates intestinal barrier function in the subject on the basis of the comparison result acquired in step S102. For the evaluation, the description thereof given for Aspect 1 is applied. The CPU 310 stores the evaluation result in the hard disk 313. The CPU 310 may output the evaluation result to allow the display section 302 to display the evaluation result, or allow a printer to print the evaluation result, or transfer the evaluation result to another computer or an application (such as a smartphone). In outputting the evaluation result, the CPU 310 may allow the display section 302 to display the measurement value for the biomarker, the reference value, and others as reference information, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone). Together with the evaluation result, the CPU 310 may allow the display section 302 to display advice on life, foods recommended to ingest (including functional foods), and others for improved intestinal barrier function, or allow a printer to print them, or transfer them to another computer or an application (such as a smartphone). Thereby, an index for evaluating intestinal barrier function in the subject can be provided, for example, to a physician, a nurse, a caregiver, a tester, a testing business operator, a service provider, or the subject himself/herself.
  • For substances selected from the substance group of the present invention, the CPU 310 evaluates intestinal barrier function in the subject on the basis of whether the comparison result acquired in step S102 satisfies the criterion shown in Table 4. Specifically, for a substance being "high" with respect to the criterion shown in Table 4, as shown in Figure 4, the CPU 310 determines whether the measurement value acquired in step 101 is higher than the reference value (i.e., whether the comparison result satisfies the criterion shown in Table 4) (step S103a); if the measurement value acquired in step 101 is higher than the reference value (i.e., the comparison result satisfies the criterion shown in Table 4), the case is evaluated as being positive (step S103b); if the measurement value acquired in step 101 is equal to or lower than the reference value (i.e., the comparison result does not satisfy the criterion shown in Table 4), the case is evaluated as being negative (step S103c). For a substance being "low" with respect to the criterion shown in Table 4, as shown in Figure 5, the CPU 310 determines whether the measurement value acquired in step 101 is lower than the reference value (i.e., whether the comparison result satisfies the criterion shown in Table 4) (step S103d); if the measurement value acquired in step 101 is lower than the reference value (i.e., the comparison result satisfies the criterion shown in Table 4), the case is evaluated as being positive (step S103e); if the measurement value acquired in step 101 is equal to or higher than the reference value (i.e., the comparison result does not satisfy the criterion shown in Table 4), the case is evaluated as being negative (step S103f).
    • < <Aspect 28> >
  • Aspect 28 of the present invention relates to a computer program for allowing a computer to execute a method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject. Hereinafter, "a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the disease or symptom".
  • The method that a computer is allowed to execute by the computer program according to Aspect 28 includes the steps of:
    • (S201) acquiring a measurement value for a biomarker in a specimen derived from the subject;
    • (S202) comparing the measurement value acquired in step S201 with a reference value to acquire a comparison result; and
    • (S203) diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject on the basis of the comparison result acquired in step S202,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 2 is applied to Aspect 28. Unless otherwise specified, the description of Aspect 27 is applied to Aspect 28. In the application, "step S101" is replaced with "step S201", "step S102" is replaced with "step S202", "step S103" is replaced with "step S203", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "diagnosing the disease or symptom in the subject", the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of diagnosing the disease or symptom in the subject", the phrase "evaluating as being positive" is replaced with the phrase "diagnosing as being positive", and the phrase "evaluating as being negative" is replaced with the phrase "diagnosing as being negative".
    • < <Aspect 29> >
  • Aspect 29 of the present invention relates to a computer program for allowing a computer to execute a method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom. Hereinafter, "a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function" may be refers to as "the risk of the development", and "probability of the subject being affected by a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the probability of being affected".
  • The method that a computer is allowed to execute by the computer program according to Aspect 29 includes the steps of:
    • (S301) acquiring a measurement value for a biomarker in a specimen derived from the subject;
    • (S302) comparing the measurement value acquired in step S301 with a reference value to acquire a comparison result; and
    • (S303) evaluating the risk or the probability on the basis of the comparison result acquired in step S302,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 4 is applied to Aspect 29. Unless otherwise specified, the description of Aspect 27 is applied to Aspect 29. In the application, "step S101" is replaced with "step S301", "step S102" is replaced with "step S302", "step S103" is replaced with "step S303", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "evaluating the risk of the development or the probability of being affected", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of evaluating the risk of the development or the probability of being affected".
    • < <Aspect 30> >
  • Aspect 30 of the present invention relates to a computer program for allowing a computer to execute a method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject. Hereinafter, "a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the disease or symptom".
  • The method that a computer is allowed to execute by the computer program according to Aspect 30 includes the steps of:
    • (S401) acquiring a measurement value for a biomarker in a specimen derived from the subject; and
    • (S402) comparing the measurement value acquired in step S401 with a reference value to acquire a comparison result as the data for diagnosing the disease or symptom caused by reduction in intestinal barrier function in the subject,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 5 is applied to Aspect 30. Unless otherwise specified, the description of Aspect 27 is applied to Aspect 30. In the application, "step S101" is replaced with "step S401", "step S102" is replaced with "step S402", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "acquiring data for diagnosing the disease or symptom in the subject", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of diagnosing the disease or symptom in the subject".
    • < <Aspect 31> >
  • Aspect 31 of the present invention relates to a computer program for allowing a computer to execute a method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function. Hereinafter, the phrase "a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the preventing or ameliorating effect on the disease or symptom".
  • The method that a computer is allowed to execute by the computer program according to Aspect 31 includes the steps of:
    • (S501) acquiring a measurement value for a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject;
    • (S502) comparing the measurement value acquired in step S501 with a reference value to acquire a comparison result; and
    • (S503) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step S502,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 6 is applied to Aspect 31. Unless otherwise specified, the description of Aspect 27 is applied to Aspect 31. In the application, "step S101" is replaced with "step S501", "step S102" is replaced with "step S502", "step S103" is replaced with "step S503", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom".
    • <<Aspect 32>>
  • Aspect 32 of the present invention relates to a computer program for allowing a computer to execute a method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function. Hereinafter, "a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function" may be referred to as "the preventing or ameliorating effect on the disease or symptom".
  • The method that a computer is allowed to execute by the computer program according to Aspect 32 includes the steps of:
    • (S601) acquiring a measurement value for a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject;
    • (S602) comparing the measurement value acquired in step S601 with a reference value to acquire a comparison result; and
    • (S603) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step S602,
    • wherein the specimen is blood, serum, or plasma, preferably plasma, and wherein the biomarker includes one or more substances selected from the substance group of the present invention.
  • Unless otherwise specified, the description of Aspect 7 is applied to Aspect 32. Unless otherwise specified, the description of Aspect 27 is applied to Aspect 32. In the application, "step S101" is replaced with "step S601", "step S102" is replaced with "step S602", "step S103" is replaced with "step S603", the phrase "evaluating intestinal barrier function in the subject" is replaced with the phrase "evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom", and the phrase "accuracy of evaluating intestinal barrier function in the subject" is replaced with the phrase "accuracy of evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom".
    • < <Aspect 33> >
  • Aspect 33 of the present invention relates to a computer-readable storage medium storing the computer program according to any one of Aspects 27 to 32.
  • Examples of the computer-readable storage medium storing the program include a non-temporary storage medium such as a ROM, a floppy disk (R), a hard disk, an optical disk, a magneto-optical disk, a CD-ROM, magnetic tape, and a nonvolatile memory card.
    • EXAMPLES
  • Hereinafter, the present invention will be described on the basis of Examples.
  • Lactulose/mannitol test, a known testing method for intestinal barrier function, was carried out for 108 test subjects. The lactulose/mannitol test was carried out as follows. Each test subject was instructed to finish his or her meal by 9 p.m. on the day before testing, and allowed to drink water before bedtime. On the day of testing, each test subject, without being allowed to eat or drink water, was subjected to sugar tolerance test. Each test subject was instructed to ingest a sugar solution (prepared by dissolving 10 g of lactulose, 5 g of mannitol, and 20 g of sucrose in 100 mL of water). During each of periods 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, and 5 to 6 hours after ingestion of the sugar solution, each test subject was instructed to ingest 150 mL or more of water. The whole of urine excreted during the period of 0 to 6 hours after ingestion of water was collected, and the amounts of lactulose and mannitol in the urine were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS was performed as follows.
  • Each measurement sample to be used for the LC-MS/MS was prepared from urine as follows. To 50 µL of urine, 400 µL of cooled acetonitrile was added, and the resultant was sufficiently stirred and left to stand on ice for 1 hour. The resulting mixture was further stirred, 800 µL of 75% by mass acetonitrile was added, the mixture was sufficiently stirred and filtered through a Millex FH 0.45-µm filter, and the resultant was used as a measurement sample. No internal standard was used.
  • Measurement by LC-MS/MS and the subsequent data analysis were performed by using a QTRAP 4500 LC-MS/MS system (Thermo Fisher Scientific). An Agilent AdvanceBio MS Spent Media 120 Å 2.1 x 100 mm (Agilent Technologies) was used as a column. The conditions for the measurement by LC-MS/MS were as follows.
    • Mobile phase A: 10 mM ammonium formate (pH 10.5)
    • Mobile phase B: acetonitrile (90%)/100 mM ammonium formate (pH 10.5) (10%)
    • Mobile condition: 0-15 min B 97%-89%, 15-18 min B 89%
    • Flow: 0.4 mL/min
    • Injection volume: 5 µL
    • Temperature: 35°C
    • IonSpray Voltage: -4500 V
  • After measuring the amounts of lactulose and mannitol in urine (hereinafter, referred to as "excretions") by LC-MS/MS, the excretion rates (%) of them were calculated with the following expression: Excretion rate = excretion (µmol/mL) × molecular weight (µg/µmol) × urine volume (mL)/amount of ingested sugar (µg) × 100.
  • After calculating the excretion rates (%), the lactulose/mannitol ratio was calculated with the following expression: Lactulose/mannitol ratio = lactulose excretion rate/mannitol excretion rate.
  • Twenty-seven test subjects with lactulose/mannitol ratios of 0.025 or more were included in a positive group, and twenty-seven test subjects with lactulose/mannitol ratios of 0.015 or less were included in a negative group.
  • Plasma and serum were obtained from the negative group and the positive group, and used in the following comparative example and example.
  • Serum was obtained as follows. On the day of the lactulose/mannitol test, 20 mL of blood was collected from each test subject, and serum was separated from part of the blood and stored in a deep freezer (-80°C) until use.
  • Plasma was obtained as follows. Prior to the sugar tolerance test conducted on the day of test, blood was collected. A portion of the blood was separated into serum and plasma, which were stored in a deep freezer (-80°C) until use.
    • < <Comparative Example 1> >
  • For each serum obtained from the negative group and the positive group, serum zonulin concentration was measured by ELISA. The ELISA was performed with a Zonuline ELISA kit (Immundiagnostik AG, Germany) in accordance with a protocol from the manufacturer.
  • An ROC (Receiver Operating Characteristic) curve was prepared by using the serum zonulin concentrations from the negative group and the positive group, and an area under the ROC curve (AUC: area under the curve) and a cutoff value were determined from the ROC curve prepared. The ROC curve, AUC, and cutoff value were determined by using the statistical analysis software "Excel Statistics" (manufactured by Social Survey Research Information Co., Ltd.). The ROC curve is a curve drawn by plotting the serum zonulin concentrations from the negative group and the positive group with an ordinate representing sensitivity and an abscissa representing (1 - specificity). The AUC is the area under the ROC curve. A point on the ROC curve at which the distance from the upper left corner of the ROC curve was minimized was employed as the cutoff value. Table 8 shows the results.
    • [Table 8]
  • Table 8
    Substance name Measurement method AUC Cutoff value
    Zonulin ELISA 0.638 39.70
    • <<Example 1>> (1) Metabolomic analysis for plasma
  • For each plasma obtained from the negative group and the positive group, metabolomic analysis was performed to comprehensively measure metabolites in the plasma. The metabolomic analysis was performed by capillary electrophoresis-Fourier transform mass spectrometry (CE-FTMS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • Each measurement sample to be used for CE-FTMS was prepared from plasma as follows. To 50 µL of plasma, 200 µL of an ethanol solution containing an internal standard (2 µM) was added, and the resultant was vortexed. To the resultant, 150 µL of Milli-Q water was added, and the mixture was stirred. A 300 µL portion of the mixture was transferred to an ultrafiltration tube, centrifuged (4°C, 9100 × g, 120 minutes), and subjected to ultrafiltration. The filtrate was dried and reconstituted in 50 µL of Milli-Q water for use as a measurement sample. The internal standard used was an internal standard (H3304-1002; HMT) from Human Metabolome Technologies, Inc. (HMT).
  • Measurement by CE-FTMS and the subsequent data analysis were performed by using an Agilent CE system (Agilent Technologies). Capillary electrophoresis was performed by using a Fused silica capillary. The conditions for the measurement by CE-FTMS were as follows.
    • [Cationic metabolites (cation mode)]
    • Run buffer: Cation Buffer Solution (p/n: H 3301-1001)
    • Rinse buffer: Cation Buffer Solution (p/n: H 3301-1001)
    • Sample injection: Pressure injection 50 mbar, 10 sec
    • CE voltage: Positive, 30 kV
    • MS ionization: ESI Positive
    • MS capillary voltage: 4,000 V
    • MS scan range: m/z 60-900
    • Sheath liquid: HMT Sheath Liquid (p/n: I 3301-1040)
    [Anionic metabolites (anion mode)]
    • Run buffer: Anion Buffer Solution (p/n: H 3302-1023)
    • Rinse buffer: Anion Buffer Solution (p/n: H 3302-1023)
    • Sample injection: Pressure injection 50 mbar, 22 sec
    • CE voltage: Positive, 30 kV
    • MS ionization: ESI Negative
    • MS capillary voltage: 3,500 V
    • MS scan range: m/z 70-1,050
    • Sheath liquid: HMT Sheath Liquid (p/n: I 3301-1040)
  • Each measurement sample to be used for LC-MS/MS was prepared from plasma as follows. Per 50 µL of plasma, 200 µL of cooled methanol containing 0.15% by mass formic acid was added, and the resultant was sufficiently stirred and left to stand on ice for 1 hour. The resulting mixture was centrifuged (12000 rpm, 10 min, 4°C), and a 200-µL portion was taken from the supernatant and brown with nitrogen gas for exsiccation, and the resultant was dissolved in 200 µL of 10% by mass methanol (containing 0.1% by mass formic acid) for measurement with an octadecylsilyl (ODS) column, or dissolved in 200 µL of 50% by mass acetonitrile for measurement with a hydrophilic interaction liquid chromatography (HILIC) column. For measurement with an ODS column, a product obtained by filtering through a GL Chromatodisk 4A aqueous 0.45-µm filter was used as a measurement sample. For measurement with a HILIC column, a product obtained by filtering through a Millex FH 0.45-µm filter was used as a measurement sample. No internal standard was used. In order to comprehensively analyze metabolites, two columns (ODS column and HILIC column) were used to detect divers substances. The ODS column is more suitable for measurement of hydrophobic substances than the HILIC column, and the HILIC column is more suitable for measurement of hydrophilic substances than the ODS column.
  • Measurement by LC-MS/MS and the subsequent data analysis were performed by using a Q Exactive Plus Orbitrab LC-MS/MS system (Thermo Fisher Scientific). A Thermo Scientific Hypersil Gold C18 (150 mm L., 2.1 mm I.D., 1.9 µm) (Thermo Fisher Scientific) was used as an ODS column, and a Merck SeQuant ZIC pHILIC (150 mm L., 2.1 mm I.D., 5 µm) (Merck Millipore) was used as a HILIC column. The conditions for the measurement by LC-MS/MS were as follows.
    • [Measurement with ODS column]
    • Mobile phase A: aqueous solution containing 0.1% by mass formic acid
    • Mobile phase B: methanol containing 0.1% by mass formic acid
    • Mobile condition: 0-3 min B 0.5%, 3-10 min B 0.5-50%, 10-12 min B 50-98%, 12-32 min B 98%, 32-33 min B 98-0.5%, 33-40 min B 0.5%
    • Flow: 0-3 min 0.1 mL/min, 3-40 min 0.25 mL/min
    • Injection volume: 2 µL
    • Temperature: 55°C
    • MS ionization: Swiching mode (ESI Positive, ESI Negative)
    • MS capillary voltage: 2.5 kV
    • MS scan range: m/z 70-1050
    [Measurement with HILIC column]
    • Mobile phase A: 10 mM aqueous solution of ammonium acetate (pH 9.6)
    • Mobile phase B: acetonitrile
    • Mobile condition: 0-3.5 min B 90%, 3.5-18 min B 90-30%, 18-21 min B 30%, 21-22 min B 30-90%, 22-30 min B 90%
    • Flow: 0.25 mL/min
    • Injection volume: 2 µL
    • Temperature: 20°C
    • MS ionization: Swiching mode (ESI Positive, ESI Negative)
    • MS capillary voltage: 2.5 kV
    • MS scan range: m/z 70-1050
  • For each plasma obtained from the negative group and the positive group, peaks derived from different substances were specified from peaks detected in CE-FTMS with reference to the m/Z values for the substances, and the peak areas for the substances were determined. The m/Z values for the substances are shown in Table 9. In determining the peak areas, only peaks with signal/noise (S/N) ratios of 3 or more were extracted from peaks detected in CE-FTMS to determine the peak areas with use of the automatic integration software MasterHands ver. 2.17.5.19 (developed by Keio University). The relative area value (relative concentration) for each substance in each plasma was determined from the following expression. Relative area value for substance in plasma = (peak area value for substance)/((peak area value for internal standard) × (mass of plasma)).
  • Plasma obtained from the negative group (27 individuals) (measurement samples No. 1 to No. 27) and plasma obtained from the positive group (27 individuals) (measurement samples No. 28 to No. 54) were sequentially subjected to measurement by LC-MS/MS. At that time, the relative area value of each substance in each plasma was calculated, with correction of the peak area value for the substance in the measurement sample on the basis of a regression curve prepared from the peak area values for the substances in a mixture (pooled sample) of plasma obtained from the negative group (27 individuals) (measurement samples No. 1 to No. 27) and plasma obtained from the positive group (27 individuals) (measurement samples No. 28 to No. 54) by using the Normalize Area node of the small molecule structure identification software Compound Discoverer 3.1 (Thermo Fisher Scientific).
  • Specifically, in addition to measurement for the measurement samples of No. 1 to No. 54, measurement for the pooled sample was performed every 15 samples, and the subsequent data analysis was then performed to determine the peak area for each substance in the pooled sample in the same manner as described above (i.e., the sequence "measurement for measurement samples of No. 1 to No. 15 -> measurement for pooled sample -> measurement for measurement samples of No. 16 to No. 30 -> measurement for pooled sample ..." was continued until completion of measurement for the sample of No. 54, and the subsequent data analysis was then performed to determine the peak area for each substance in the pooled sample in the same manner as described above). The relative area value of each substance in each plasma was calculated with correction of the peak area value for the substance in the measurement sample in the same manner as described above.
  • The correction with use of the pooled sample was carried out for the purpose of correcting errors, for example, caused by the influence of the temporal attenuation of the peak area values for the substances.
  • An ROC (Receiver Operating Characteristic) curve was prepared with use of the relative area values for each substance in the plasma, and an area under the ROC curve (AUC: area under the curve) and a cutoff value were determined from the ROC curve prepared. The ROC curve, AUC, and cutoff value were determined by using the statistical analysis software "Excel Statistics" (manufactured by Social Survey Research Information Co., Ltd.). The ROC curve is a curve drawn by plotting the relative area values for a substance in the plasma with an ordinate representing sensitivity and an abscissa representing (1 - specificity). The AUC is the area under the ROC curve. A point on the ROC curve at which the distance from the upper left corner of the ROC curve was minimized was employed as the cutoff value. Table 9 shows the results. Values of AUC closer to 1 indicate higher accuracy of evaluating intestinal barrier function. Cases with a value of AUC of 0.6 or less can be classified as low evaluation accuracy, cases with a value of AUC of more than 0.6 and 0.7 or less as slightly high evaluation accuracy, cases with a value of AUC of more than 0.7 and 0.8 or less as high evaluation accuracy, and cases with a value of AUC of more than 0.8 as very high evaluation accuracy.
  • Among the substances comprehensively measured, Table 9 shows results only for substances with AUC of 0.65 or more (the substances of No. 1 to No. 42 in the substance group of the present invention).
  • For each of the substances of No. 1 to No. 42 in the substance group of the present invention, the positive group average value, the negative group average value, and the ratio of the positive group average value to the negative group average value (positive group average value / negative group average value) were determined by using the relative area values for the substance in the plasma. Table 10 shows the results. Table 10 also shows tendencies in the positive group. The entry "increase" indicates the presence of a tendency to increase in the positive group, and the entry "decrease" indicates the presence of a tendency to decrease in the positive group.
  • The results shown in Tables 9 and 10 revealed that in the case of a specimen of plasma, intestinal barrier function in a subject can be evaluated with high accuracy by using one or more substances selected from the substance group of the present invention as a biomarker. As AUC closer to 1 indicates higher accuracy of evaluating intestinal barrier function, the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide) and the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine) give particularly high accuracy of evaluating intestinal barrier function. [Table 9-1]
    Table 9 (Substance group of present invention)
    No. Substance name Measurement method m/z AUC Cutoff value
    1 Melamine CE-FTMS 127.073 0.7791 2.864E-04
    2 Azelaic acid CE-FTMS 187.097 0.7366 1.070E-04
    3 Urocanic acid CE-FTMS 139.050 0.7188 4.142E-04
    4 Acetanilide CE-FTMS 136.076 0.7133 2.701E-04
    5 Paraxanthine CE-FTMS 181.072 0.7078 1.032E-02
    6 4-Acetamidobutanoic acid CE-FTMS 144.066 0.7078 5.627E-05
    7 Glycerol CE-FTMS 93.055 0.7064 8.691E-02
    8 N-Acetylputrescine CE-FTMS 131.118 0.7010 1.768E-04
    9 N8-Acetylspermidine CE-FTMS 188.176 0.6996 7.304E-05
    10 Threonine CE-FTMS 120.066 0.6968 1.428E-01
    11 Aminophylline CE-FTMS 181.072 0.6927 1.691E-03
    12 2-Oxooctanoic acid CE-FTMS 157.087 0.6790 8.658E-05
    13 Cyclohexanecarboxylic acid CE-FTMS 127.076 0.6749 1.000E-07
    14 Muscimol CE-FTMS 115.050 0.6749 1.386E-04
    15 3-Methoxytyrosine CE-FTMS 212.092 0.6708 2.340E-04
    16 Theobromine CE-FTMS 181.072 0.6694 8.347E-03
    17 Methionine sulfoxide CE-FTMS 166.053 0.6653 2.576E-03
    18 Glyoxylic acid CE-FTMS 72.993 0.6639 2.699E-05
    19 Serotonin CE-FTMS 177.102 0.6626 2.369E-04
    20 Histidylglutamic acid CE-FTMS 285.119 0.6591 1.206E-05
    21 Guanidinosuccinic acid CE-FTMS 176.067 0.6571 7.367E-04
    22 Uracil CE-FTMS 113.034 0.6557 2.266E-03
    23 Ergothioneine CE-FTMS 230.096 0.6529 1.044E-05
    24 Betonicine CE-FTMS 160.097 0.6447 1.000E-07
    25 Adenine CE-FTMS 136.062 0.6324 1.592E-05
    [Table 9-2]
    Table 9 (Continued)
    No. Substance name Measurement method m/z AUC Cutoff value
    26 N-Methylnicotinamide CE-FTMS 137.071 0.6111 1.844E-06
    27 4-Hexyloxyaniline LC-MS/MS 193.147 0.7407 1.199E+07
    28 Caffeine LC-MS/MS 194.081 0.7202 8.215E+05
    29 Theophylline LC-MS/MS 180.065 0.7037 6.539E+07
    30 DL-β-Leucine LC-MS/MS 131.095 0.6941 4.635E+06
    31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide LC-MS/MS 246.137 0.6900 8.709E+05
    32 Guanidineacetic acid LC-MS/MS 117.054 0.6886 2.448E+07
    33 Acetaminophen glucuronide LC-MS/MS 327.096 0.6859 1.841E+05
    34 Propyphenazone LC-MS/MS 230.142 0.6859 5.845E+04
    35 L-Threonic acid LC-MS/MS 136.038 0.6859 1.615E+08
    36 2-Ethylamino-1-phenylpropanol LC-MS/MS 179.131 0.6845 1.575E+05
    37 2-Hydroxyhippuric acid LC-MS/MS 195.053 0.6831 1.374E+05
    38 4-Dodecylbenzenesulfonic acid LC-MS/MS 326.193 0.6790 8.986E+07
    39 Uridine LC-MS/MS 244.070 0.6790 2.483E+08
    40 Ofloxacin LC-MS/MS 361.143 0.6722 1.152E+05
    41 (2R)-2,3-Dihydroxypropanoic acid LC-MS/MS 106.027 0.6694 9.356E+06
    42 Lactamide LC-MS/MS 89.048 0.6598 2.050E+08
    [Table 10-1]
    Table 10 (Substance group of present invention)
    No. Substance name Tendency in positive group Negative group average value Positive group average value Positive group average value / Negative group average value
    1 Melamine Decrease 3.168E-04 2.178E-04 0.687
    2 Azelaic acid Increase 8.195E-05 1.557E-04 1.900
    3 Urocanic acid Decrease 6.237E-04 4.095E-04 0.657
    4 Acetanilide Decrease 2.833E-04 1.773E-04 0.626
    5 Paraxanthine Decrease 1.993E-02 9.842E-03 0.494
    6 4-Acetamidobutanoic acid Decrease 6.034E-05 4.661E-05 0.773
    7 Glycerol Increase 8.784E-02 1.060E-01 1.207
    8 N-Acetylputrescine Decrease 2.073E-04 1.675E-04 0.808
    9 N8-Acetylspermidine Decrease 9.601E-05 7.645E-05 0.796
    10 Threonine Decrease 1.584E-01 1.412E-01 0.892
    11 Aminophylline Decrease 2.912E-03 1.602E-03 0.550
    12 2-Oxooctanoic acid Decrease 1.336E-04 9.282E-05 0.695
    13 Cyclohexanecarboxylic acid Decrease 1.992E-05 5.009E-06 0.251
    14 Muscimol Decrease 1.528E-04 1.287E-04 0.842
    15 3-Methoxytyrosine Decrease 2.603E-04 2.276E-04 0.874
    16 Theobromine Decrease 1.492E-02 9.816E-03 0.658
    17 Methionine sulfoxide Decrease 3.788E-03 3.149E-03 0.831
    18 Glyoxylic acid Decrease 2.977E-05 2.272E-05 0.763
    19 Serotonin Increase 2.219E-04 2.781E-04 1.253
    20 Histidylglutamic acid Decrease 1.215E-05 9.876E-06 0.813
    21 Guanidinosuccinic acid Increase 7.915E-04 1.121E-03 1.417
    22 Uracil Increase 2.216E-03 2.519E-03 1.136
    23 Ergothioneine Increase 8.950E-07 4.378E-04 489.207
    24 Betonicine Decrease 2.238E-04 1.293E-05 0.058
    [Table 10-2]
    Table 10 (Continued)
    No. Substance name Tendency in positive group Negative group average value Positive group average value Positive group average value / Negative group average value
    25 Adenine Increase 7.501E-07 7.279E-06 9.705
    26 N-Methylnicotinamide Increase 0 1.301E-06 -
    27 4-Hexyloxyaniline Increase 1.157E+07 1.541E+07 1.333
    28 Caffeine Decrease 4.364E+06 2.741E+06 0.628
    29 Theophylline Decrease 1.164E+08 6.455E+07 0.555
    30 DL-β-Leucine Increase 8.789E+06 9.978E+06 1.135
    31 N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide Increase 9.144E+05 2.826E+07 30.909
    32 Guanidineacetic acid Decrease 3.252E+07 2.661E+07 0.818
    33 Acetaminophen glucuronide Increase 1.649E+05 4.137E+06 25.083
    34 Propyphenazone Increase 4.905E+04 2.995E+07 610.573
    35 L-Threonic acid Decrease 1.793E+08 1.541E+08 0.859
    36 2-Ethylamino-1-phenylpropanol Increase 1.457E+05 2.026E+06 13.899
    37 2-Hydroxyhippuric acid Increase 2.911E+05 3.086E+05 1.060
    38 4-Dodecylbenzenesulfonic acid Decrease 1.339E+08 1.069E+08 0.799
    39 Uridine Increase 2.167E+08 2.522E+08 1.164
    40 Ofloxacin Increase 7.943E+04 1.035E+05 1.303
    41 (2R)-2,3-Dihydroxypropanoic acid Decrease 1.068E+07 8.779E+06 0.822
    42 Lactamide Decrease 2.337E+08 2.081E+08 0.891
    • (2) Combined variable (statistical analysis value) (2-1) Substances of No. 1 to No. 4 in the substance group of the present invention
  • For two or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention (melamine, azelaic acid, urocanic acid, and acetanilide), a combined variable was determined by using the relative area values obtained in (1) above with an expression shown below, an ROC curve was prepared on the basis of the combined variable determined, and AUC and a cutoff value were determined from the ROC curve prepared. Table 11 shows the results. The coefficients and constant term were determined through multinomial logistic regression analysis. The coefficients and constant term are as shown in Table 2. In Table 11, "CP_A" refers to melamine, "CP_B" refers to azelaic acid, "CP_C" refers to urocanic acid, and "CP_D" refers to acetanilide. Statistical analysis value = (Coefficient 1 × (Concentration of first substance)) + (Coefficient 2 × (Concentration of second substance)) +...+ (Coefficient n × (Concentration of n-th substance)) + Constant term
  • As demonstrated in Table 11, it was revealed that an ROC curve with increased AUC is successfully given, that is, enhanced accuracy of evaluating intestinal barrier function is successfully achieved by combining two or more substances selected from the substances of No. 1 to No. 4 in the substance group of the present invention. [Table 11]
    Table 11 (No. 1 to No. 4 in substance group of present invention)
    Number of substances combined First substance Second substance Third substance Fourth substance AUC Cutoff value
    2 CP_A CP_B - - 0.798 0.4474
    2 CP_A CP_C - - 0.842 -0.0782
    2 CP_A CP_D - - 0.771 0.1073
    2 CP_B CP_C - - 0.841 -0.1443
    2 CP_B CP_D - - 0.786 0.1026
    2 CP_C CP_D - - 0.811 0.3187
    3 CP_A CP_B CP_C - 0.886 -0.4402
    3 CP_A CP_B CP_D - 0.807 0.4774
    3 CP_A CP_C CP_D - 0.842 -0.0491
    3 CP_B CP_C CP_D - 0.848 0.0037
    4 CP_A CP_B CP_C CP_D 0.901 0.0812
    • (2-2) Substances of No. 27 to No. 30 in the substance group of the present invention
  • For two ir more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention (4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine), a combined variable was determined by using the relative area values obtained in (1) above in the same manner as in (2-1) above, an ROC curve was prepared on the basis of the combined variable determined, and AUC and a cutoff value were determined from the ROC curve prepared. Table 12 shows the results. The coefficients and constant term were determined through multinomial logistic regression analysis. The coefficients and constant term are as shown in Table 3. In Table 12, "LP_A" refers to 4-hexyloxyaniline, "LP_B" refers to caffeine, "LP_C" refers to theophylline, and "LP_D" refers to DL-β-leucine.
  • As demonstrated in Table 12, it was revealed that an ROC curve with increased AUC is successfully given, that is, enhanced accuracy of evaluating intestinal barrier function is successfully achieved by combining two or more substances selected from the substances of No. 27 to No. 30 in the substance group of the present invention. [Table 12]
    Table 12 (No. 27 to No. 30 in substance group of present invention)
    Number of substances combined First substance Second substance Third substance Fourth substance AUC Cutoff value
    2 LP_A LP_B - - 0.761 0.2267
    2 LP_A LP_C - - 0.752 0.2174
    2 LP_A LP_D - - 0.739 0.0855
    2 LP_B LP_C - - 0.711 -0.1752
    2 LP_B LP_D - - 0.730 -0.2033
    2 LP_C LP_D - - 0.702 -0.2041
    3 LP_A LP_B LP_C - 0.762 -0.0585
    3 LP_A LP_B LP_D - 0.769 0.0697
    3 LP_A LP_C LP_D - 0.761 0.1699
    3 LP_B LP_C LP_D - 0.699 -0.0373
    4 LP_A LP_B LP_C LP_D 0.760 0.1397

Claims (50)

  1. A method for evaluating intestinal barrier function in a subject, the method comprising the steps of:
    (1a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
    (1b) comparing the measurement value acquired in step 1a with a reference value to acquire a comparison result; and
    (1c) evaluating intestinal barrier function in the subject on the basis of the comparison result acquired in step 1b,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the following group consisting of:
    melamine;
    azelaic acid;
    urocanic acid;
    acetanilide;
    paraxanthine;
    4-acetamidobutanoic acid;
    glycerol;
    N-acetylputrescine;
    N8-acetylspermidine;
    threonine;
    aminophylline;
    2-oxooctanoic acid;
    cyclohexanecarboxylic acid;
    muscimol;
    3-methoxytyrosine;
    theobromine;
    methionine sulfoxide;
    glyoxylic acid;
    serotonin;
    histidylglutamic acid;
    guanidinosuccinic acid;
    uracil;
    ergothioneine;
    betonicine;
    adenine;
    N-methylnicotinamide;
    4-hexyloxyaniline;
    caffeine;
    theophylline;
    DL-β-leucine;
    N1-[4-(acetylamino)phenyl]-2,2-dimethylcyclopropane-1-carboxamide;
    guanidineacetic acid;
    acetaminophen glucuronide;
    propyphenazone;
    L-threonic acid;
    2-ethylamino-1-phenylpropanol;
    2-hydroxyhippuric acid;
    4-dodecylbenzenesulfonic acid;
    uridine;
    ofloxacin;
    (2R)-2,3-dihydroxypropanoic acid; and
    lactamide.
  2. A method for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, the method comprising the steps of:
    (2a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
    (2b) comparing the measurement value acquired in step 2a with a reference value to acquire a comparison result; and
    (2c) diagnosing the disease or symptom on the basis of the comparison result acquired in step 2b,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  3. A method for treating a disease or symptom caused by reduction in intestinal barrier function in a subject, the method comprising the steps of:
    (3a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
    (3b) comparing the measurement value acquired in step 3a with a reference value to acquire a comparison result;
    (3c) diagnosing the disease or symptom on the basis of the comparison result acquired in step 3b; and
    (3d) administering a substance or composition for treating the disease or symptom to the subject if the subject is diagnosed to be affected by the disease or symptom,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  4. A method for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom, the method comprising the steps of:
    (4a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker;
    (4b) comparing the measurement value acquired in step 4a with a reference value to acquire a comparison result; and
    (4c) evaluating the risk or the probability on the basis of the comparison result acquired in step 4b,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  5. A method for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject, the method comprising the steps of:
    (5a) measuring a biomarker in a specimen derived from the subject to acquire a measurement value for the biomarker; and
    (5b) comparing the measurement value acquired in step 5a with a reference value to acquire a comparison result as the data,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  6. A method for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the method comprising the steps of:
    (6a) administering a candidate substance or candidate composition to a subject having reduced intestinal barrier function;
    (6b) measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker;
    (6c) comparing the measurement value acquired in step 6b with a reference value to acquire a comparison result; and
    (6d) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 6c,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  7. A method for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the method comprising the steps of:
    (7a) administering the candidate substance or candidate composition to a subject having reduced intestinal barrier function;
    (7b) measuring a biomarker in a specimen obtained from the subject after administration of the candidate substance or candidate composition to acquire a measurement value for the biomarker;
    (7c) comparing the measurement value acquired in step 7b with a reference value to acquire a comparison result; and
    (7d) evaluating the candidate substance or candidate composition for the improving effect on intestinal barrier function or the preventing or ameliorating effect on the disease or symptom on the basis of the comparison result acquired in step 7c,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  8. The method according to any one of claims 1 to 7, wherein the intestinal barrier function is barrier function of a small intestine.
  9. The method according to any one of claims 1 to 7, wherein the biomarker comprises two, three, or four or more substances selected from the group.
  10. The method according to any one of claims 1 to 7, wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
  11. The method according to claim 10, wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
  12. The method according to any one of claims 1 to 7, wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
  13. The method according to claim 12, wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
  14. A biomarker in a specimen derived from a subject for use in evaluating intestinal barrier function in the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  15. A biomarker in a specimen derived from a subject for use in diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  16. A biomarker in a specimen derived from a subject for use in evaluating the subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  17. A biomarker in a specimen derived from a subject for use in acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  18. A biomarker in a specimen for use in screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  19. A biomarker in a specimen for use in evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  20. The biomarker according to any one of claims 14 to 19, wherein the intestinal barrier function is barrier function of a small intestine.
  21. The biomarker according to any one of claims 14 to 19, wherein the biomarker comprises two, three, or four or more substances selected from the group.
  22. The biomarker according to any one of claims 14 to 19, wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
  23. The biomarker according to claim 22, wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
  24. The biomarker according to any one of claims 14 to 19, wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
  25. The biomarker according to claim 24, wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
  26. Use of a biomarker in a specimen derived from a subject for evaluating intestinal barrier function in the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  27. Use of a biomarker in a specimen derived from a subject for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  28. Use of a biomarker in a specimen derived from a subject for evaluating the subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  29. Use of a biomarker in a specimen derived from a subject for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  30. Use of a biomarker in a specimen for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  31. Use of a biomarker in a specimen for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function, the specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  32. The use according to any one of claims 26 to 31, wherein the intestinal barrier function is barrier function of a small intestine.
  33. The use according to any one of claims 26 to 31, wherein the biomarker comprises two, three, or four or more substances selected from the group.
  34. The use according to any one of claims 26 to 31, wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
  35. The use according to claim 34, wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
  36. The use according to any one of claims 26 to 31,wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
  37. The use according to claim 36, wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
  38. A kit for evaluating intestinal barrier function in a subject,
    wherein the kit comprises one or more reagents for measuring a biomarker in a specimen derived from the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  39. A kit for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject,
    wherein the kit comprises one or more reagents for measuring a biomarker in a specimen derived from the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  40. A kit for evaluating a subject's risk of developing a disease or symptom caused by reduction in intestinal barrier function, or probability of the subject being affected by the disease or symptom,
    wherein the kit comprises one or more reagents for measuring a biomarker in a specimen derived from the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  41. A kit for acquiring data for diagnosing a disease or symptom caused by reduction in intestinal barrier function in a subject,
    wherein the kit comprises one or more reagents for measuring a biomarker in a specimen derived from the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  42. A kit for screening for a substance or composition having an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function,
    wherein the kit comprises one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of a candidate substance or candidate composition to the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  43. A kit for evaluating a candidate substance or candidate composition for an improving effect on intestinal barrier function or a preventing or ameliorating effect on a disease or symptom caused by reduction in intestinal barrier function,
    wherein the kit comprises one or more reagents for measuring a biomarker in a specimen obtained from a subject having reduced intestinal barrier function after administration of the candidate substance or candidate composition to the subject,
    wherein the specimen is blood, serum, or plasma, and
    wherein the biomarker comprises one or more substances selected from the group specified in claim 1.
  44. The kit according to any one of claims 38 to 43, wherein the intestinal barrier function is barrier function of a small intestine.
  45. The kit according to any one of claims 38 to 43, wherein the biomarker comprises two, three, or four or more substances selected from the group.
  46. The kit according to any one of claims 38 to 43, wherein the biomarker comprises one or more substances selected from the group consisting of melamine, azelaic acid, urocanic acid, and acetanilide.
  47. The kit according to claim 46, wherein the biomarker in the specimen is measured by capillary electrophoresis-mass spectrometry.
  48. The kit according to any one of claims 38 to 43, wherein the biomarker comprises one or more substances selected from the group consisting of 4-hexyloxyaniline, caffeine, theophylline, and DL-β-leucine.
  49. The kit according to claim 48, wherein the biomarker in the specimen is measured by liquid chromatography-mass spectrometry.
  50. A method for producing a food product or pharmaceutical composition, the method comprising the step of blending a substance or composition obtained through screening by the method according to claim 6 with one or two or more components to constitute the food product or pharmaceutical composition.
EP24770772.2A 2023-03-10 2024-03-08 Biomarker and use thereof Pending EP4679085A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2023038050 2023-03-10
PCT/JP2024/009046 WO2024190666A1 (en) 2023-03-10 2024-03-08 Biomarker and use thereof

Publications (1)

Publication Number Publication Date
EP4679085A1 true EP4679085A1 (en) 2026-01-14

Family

ID=92755620

Family Applications (1)

Application Number Title Priority Date Filing Date
EP24770772.2A Pending EP4679085A1 (en) 2023-03-10 2024-03-08 Biomarker and use thereof

Country Status (4)

Country Link
EP (1) EP4679085A1 (en)
JP (1) JPWO2024190666A1 (en)
CN (1) CN120883053A (en)
WO (1) WO2024190666A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025205714A1 (en) * 2024-03-26 2025-10-02 株式会社明治 Target bacteria and use therefor

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015168405A1 (en) * 2014-05-02 2015-11-05 The Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and materials for characterizing intestinal barrier function
EP3201622A4 (en) * 2014-10-03 2018-05-30 The University of Ottawa Markers for inflammatory bowel disease
WO2017210751A1 (en) * 2016-06-10 2017-12-14 Newsouth Innovations Pty Limited Diagnostic method
EP3884062B1 (en) * 2018-11-22 2022-09-21 Universiteit Gent Use of bacterial extracellular vesicles as a biomarker for intestinal barrier permeability and microbial dysbiosis

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ANN TRANSL MED, vol. 7, no. 16, 2019, pages 1 - 10, Retrieved from the Internet <URL:http://dx.doi.org/10.21037/atm.2019.07.102>
ARISTO VOJDANI ET AL.: "Fluctuation of zonulin levels in blood vs stability of antibodies.", WORLD JOURNAL OF GASTROENTEROLOGY., vol. 23, no. 31, 2017, pages 5669 - 5679
FASANA A ET AL.: "Zonulin, ''a newly discovered modulator of intestinal permeability, and its expression in coeliac disease", THE LANCET., vol. 355, 2000, pages 1518 - 1519
METAB BRAIN DIS, 11 May 2017 (2017-05-11)
PNAS, vol. 100, no. 21, 14 October 2003 (2003-10-14), pages 12343 - 12348, Retrieved from the Internet <URL:www.pnas.org/cgi/doi/10.1073/pnas.2033602100>
See also references of WO2024190666A1

Also Published As

Publication number Publication date
WO2024190666A1 (en) 2024-09-19
CN120883053A (en) 2025-10-31
JPWO2024190666A1 (en) 2024-09-19

Similar Documents

Publication Publication Date Title
Schaub et al. SGLT2 inhibitors mitigate kidney tubular metabolic and mTORC1 perturbations in youth-onset type 2 diabetes
Laurans et al. Genetic deficiency of indoleamine 2, 3-dioxygenase promotes gut microbiota-mediated metabolic health
Pacifico et al. Acylated and nonacylated ghrelin levels and their associations with insulin resistance in obese and normal weight children with metabolic syndrome
Van Langenberg et al. Delving into disability in Crohn's disease: dysregulation of molecular pathways may explain skeletal muscle loss in Crohn's disease
Dalton et al. Gut permeability in autism spectrum disorders
Chen et al. d-Ribose as a Contributor to Glycated Haemoglobin
Søfteland et al. Pancreatic exocrine insufficiency in diabetes mellitus-prevalence and characteristics
Qu et al. Plasma fetuin-B concentrations are associated with insulin resistance and first-phase glucose-stimulated insulin secretion in individuals with different degrees of glucose tolerance
Hong et al. Angiopoietin‐like protein 8/betatrophin correlates with hepatocellular lipid content independent of insulin resistance in non‐alcoholic fatty liver disease patients
Dickinson et al. Effect of renal or hepatic impairment on the pharmacokinetics of mirabegron
US20150247839A1 (en) Cmpf as a biomarker for diabetes and associated methods
Chatzianagnostou et al. Low concordance between HbA1c and OGTT to diagnose prediabetes and diabetes in overweight or obesity
EP4679085A1 (en) Biomarker and use thereof
Maurer et al. Renal function is independently associated with circulating betatrophin
Tang et al. Dietary galacto-oligosaccharides ameliorate atopic dermatitis-like skin inflammation and behavioral deficits by modulating gut microbiota–brain–skin axis
Mastrototaro et al. The role of exosomes for sustained specific cardiorespiratory and metabolic improvements in males with type 2 diabetes after detraining
Lv et al. Microbial imidazole propionate affects glomerular filtration rate in patients with diabetic nephropathy through association with HSP90α
EP2435826B1 (en) Urinary gm2 activator protein as a marker of acute renal failure or the risk of developing acute renal failure
US10690682B2 (en) Method for the diagnosis of metachromatic leukodystrophy
Rodriguez et al. Late-onset renal hypertrophy and dysfunction in mice lacking CTRP1
EP4679086A1 (en) Biomarker and use thereof
Kirchhoff et al. Impact of different fat depots on insulin sensitivity: predominant role of liver fat
Tomassini et al. 1H NMR-based urinary metabolic profiling reveals changes in nicotinamide pathway intermediates due to postnatal stress model in rat
EP4679087A1 (en) Biomarker and use thereof
Lisowska-Myjak et al. Serum indoxyl sulphate and its relation to albumin and α1-acid glycoprotein as a potential biomarkers of maternal intestinal metabolism during pregnancy and postpartum

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20251009

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR